3D printed ceramic compositions and methods of use

Abstract

The present disclosure provides devices comprising a therapeutic agent bound to a printed three-dimensional structure. Ink formulations for three-dimensional printing are also disclosed. Further provided herein are methods for making the devices and uses thereof, such as for treating disease in a subject in need thereof.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 63 / 373,278, filed August 23, 2022, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing, which is hereby submitted in XML format and is incorporated by reference in its entirety. The XML copy, created on August 22, 2023, is named 50222-711_601.xml and is 721,461 bytes in size. [Background technology]

[0003] Three-dimensional (3D) printing is a manufacturing process that creates three-dimensional solid objects from digital files. The additive process of 3D printing generates objects with precision up to micrometers by successively adding layers of material until the desired object is created. When combined with computer-aided design (CAD) software, 3D printing enables the production of complex functional shapes that can be easily customized compared to traditional manufacturing methods.

[0004] Surgical implantation of scaffolds and / or other forms of implantable materials to promote tissue regeneration is a useful technique if the implant can match the mechanical properties of natural tissue. Various materials, including those that mimic tissue and allow tissue regrowth, can be used as inks in the fabrication of porous 3D-printed structures for implantation. Inks with effective bioactive and mechanical properties are necessary for natural tissue regeneration, and when used in 3D printing, they can be customized for the repair of large tissue defects. Summary of the Invention

[0005] The present disclosure provides ink formulations and methods for 3D printing scaffolds. Additionally, scaffolds that can be produced using such ink formulations and methods are provided. The scaffolds may be coated with therapeutic agents, such as those that promote bone growth, and / or seeded with cells to produce devices for use in tissue replacement and transplantation. In some devices, therapeutic agents may be tethered to the scaffold via targeting moieties that interact with scaffold components, such as ceramic materials. Advantages of the materials and methods described herein include the creation of customizable 3D-printed implants, as well as more general-purpose objects, such as strip-shaped, block-shaped, or cylindrical objects. Because the implants are 3D printed, precise control of the implant shape is possible. Therefore, implants printed with these inks are suitable for a variety of treatments, such as long bone repair, spinal fusion, and maxillofacial structures. Different ink formulations can result in implantable devices with different properties, such as porosity and flexibility.

[0006] Various scaffolds of the present disclosure are prepared using fused granule (FGF) 3D printing. Inks for use with such FGF 3D printing methods can be designed with specific viscosities that minimize oozing of the molten ink from the 3D printer nozzle during non-printing operations, yet are low enough to allow flow through nozzles with diameters of several hundred microns when the screw extruder is activated for printing operations. In a non-limiting example, the ink comprises one or more water-soluble polymers, the identity and / or molecular weight of which can be increased or decreased to adjust the viscosity of the ink. For example, the one or more water-soluble polymers can comprise polyethylene glycol (PEG), with higher molecular weight PEGs resulting in higher melt viscosities. Non-limiting examples of inks are described in the Examples section of this specification.

[0007] Various inks and scaffolds of the present disclosure are designed to improve the accessibility of therapeutic agents and / or targeting moieties to scaffold components, such as ceramic materials like β-tricalcium phosphate. For example, ink formulations containing sacrificial pore-forming agents. Sacrificial pore-forming agents include water-soluble polymers such as PEG, as well as the microparticles described herein. Non-limiting examples of inks containing sacrificial pore-forming agents are described in the Examples section of this specification.

[0008] The various inks and methods disclosed herein are designed to improve the manufacturability of 3D printed scaffolds by converting the ink into pellets. Pellets are a common form of polymer feedstock that allows for easy storage and transportation on a large scale. Pellets do not require stringent mechanical properties or dimensional tolerances (compared to 3D printing filaments). Pellets can be continuously supplied on a large scale via a hopper, as opposed to a finite spool of filament that must be periodically replaced.

[0009] The various inks and scaffolds disclosed herein are designed to optimize the bioabsorption properties of the scaffold. For example, copolymers used in certain formulations, such as caprolactone / glycolide copolymer (95:5), caprolactone / glycolide copolymer (90:10), and poly(D,L-lactide-co-glycolide) copolymer (50:50), have faster absorption rates than other formulations, such as polycaprolactone. Absorption rates range from slowest to fastest, such as polycaprolactone, polycaprolactone / polyglycolide copolymer (95:5), polycaprolactone / glycolide copolymer (90:10), polydioxanone / L-lactide copolymer (90:10), and poly(D,L-lactide-co-glycolide) copolymer (50:50).

[0010] In one aspect, provided herein is an ink formulation comprising about 55% to about 65% by weight of β-tricalcium phosphate (βTCP), about 15% to about 25% by weight of caprolactone / glycolide copolymer, about 5% to about 15% of polyethylene glycol (PEG) having a molecular weight of about 500 g / mol to about 15,000 g / mol, and about 5% to about 15% of PEG having a molecular weight of about 25,000 g / mol to about 50,000 g / mol. In some embodiments, the ink formulation comprises about 60% by weight of βTCP, about 20% by weight of caprolactone / glycolide copolymer, about 10% by weight of PEG having a molecular weight of about 500 g / mol to about 15,000 g / mol, and about 10% of PEG having a molecular weight of about 25,000 g / mol to about 50,000 g / mol. In some embodiments, the ink formulation comprises about 60% by weight β-TCP, about 20% by weight caprolactone / glycolide copolymer, about 10% by weight PEG having a molecular weight of about 8,000 g / mol, and about 10% by weight PEG having a molecular weight of about 35,000 g / mol. In some embodiments, the caprolactone / glycolide copolymer is a 95:5 caprolactone / glycolide copolymer. In some embodiments, the caprolactone / glycolide copolymer is a 90:10 caprolactone / glycolide copolymer.

[0011] In some embodiments, provided herein is a method for preparing a three-dimensional structure, the method comprising additive manufacturing using an ink formulation. In some embodiments, the ink formulation is in the form of pellets. In some embodiments, the additive manufacturing comprises fused granulation (FGF).

[0012] In some embodiments, provided herein are structures prepared by additive manufacturing of ink formulations.

[0013] In some embodiments, provided herein is a method of treating a bone defect in a subject in need thereof, the method comprising applying a structure to the bone defect in the subject. In some embodiments, the defect is in the spine.

[0014] In some embodiments, provided herein are devices comprising a therapeutic agent and a structure. In some embodiments, the therapeutic agent is non-covalently bound to the structure. In some embodiments, the therapeutic agent comprises a growth factor. In some embodiments, the growth factor is selected from Table 1. In some embodiments, the therapeutic agent comprises a bone morphogenetic protein (BMP). In some embodiments, the therapeutic agent comprises a targeting moiety, and the targeting moiety is non-covalently bound to the structure. In some embodiments, the targeting moiety comprises a polypeptide at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to any one of the sequences in Tables 2-3. In some embodiments, the therapeutic agent comprises a chimeric polypeptide comprising a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to any one of SEQ ID NOs: 433-441.

[0015] In some embodiments, provided herein is a method of treating a bone defect in a subject in need thereof, the method comprising applying a device to the bone defect in the subject. In some embodiments, the defect is in the spine.

[0016] In one aspect, provided herein is a three-dimensional structure comprising 70% to about 80% by weight of β-TCP and about 20% to about 30% by weight of polycaprolactone (PCL). In some embodiments, the three-dimensional structure comprises about 75% by weight of β-TCP and about 25% by weight of PCL. In one aspect, provided herein is a three-dimensional structure comprising about 70% to about 80% by weight of β-TCP and about 20% to about 30% by weight of caprolactone / glycolide copolymer. In some embodiments, the three-dimensional structure comprises about 75% by weight of β-TCP and about 25% by weight of caprolactone / glycolide copolymer. In some embodiments, the caprolactone / glycolide copolymer is a 95:5 caprolactone / glycolide copolymer. In some embodiments, the caprolactone / glycolide copolymer is a 90:10 caprolactone / glycolide copolymer. In one aspect, provided herein is a three-dimensional structure comprising about 70% to about 80% by weight of β-TCP and about 20% to about 30% by weight of poly(D,L-lactide-co-glycolide) copolymer. In some embodiments, the three-dimensional structure comprises about 75% by weight of β-TCP and about 25% by weight of poly(D,L-lactide-co-glycolide) copolymer. In some embodiments, the caprolactone / glycolide copolymer is poly(D,L-lactide-co-glycolide) copolymer (50:50). In one aspect, provided herein is a three-dimensional structure comprising about 70% to about 80% by weight of β-TCP and about 20% to about 30% by weight of polydioxanone (PDS). In some embodiments, the three-dimensional structure comprises about 75% by weight of β-TCP and about 25% by weight of PDS. In one aspect, provided herein is a three-dimensional structure comprising about 70% to about 80% by weight of β-TCP and about 20% to about 30% by weight of dioxanone / L-lactide copolymer. In some embodiments, the three-dimensional structure comprises about 75% by weight of β-TCP and about 25% by weight of dioxanone / L-lactide copolymer. In some embodiments, the caprolactone / glycolide copolymer is a dioxanone / L-lactide copolymer (90:10).In one aspect, provided herein is a three-dimensional structure comprising about 70% to about 80% by weight of β-TCP and about 20% to about 30% by weight of glycolide / L-lactide copolymer. In some embodiments, the three-dimensional structure comprises about 75% by weight of β-TCP and about 25% by weight of glycolide / L-lactide copolymer. In some embodiments, the caprolactone / glycolide copolymer is glycolide / L-lactide copolymer (95:5). In one aspect, provided herein is a three-dimensional structure comprising about 70% to about 80% by weight of β-TCP and about 20% to about 30% by weight of poly-L-lactide. In some embodiments, the three-dimensional structure comprises about 75% by weight of β-TCP and about 25% by weight of poly-L-lactide.

[0017] In some embodiments of the three-dimensional structures herein, the density is about 1 g / cm to 1.5 g / cm. In some embodiments of the three-dimensional structures herein, the open porosity is about 25% to about 40%. In some embodiments of the three-dimensional structures herein, the strut diameter is about 300 μm to 800 μm, or about 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, or 800 μm. In some embodiments of the three-dimensional structures herein, the structure comprises a plurality of micropores, and the average pore size of the micropores is about 1 micron to about 500 microns, or about 1 micron to about 50 microns.

[0018] Further provided herein are methods for preparing the three-dimensional structures, the methods comprising additive manufacturing. In some embodiments, the additive manufacturing comprises fused granule manufacturing (FGF) or fused filament manufacturing (FFF).

[0019] Further provided is a device comprising the structure provided herein and a therapeutic agent. In some embodiments, the therapeutic agent is non-covalently bound to the structure. In some embodiments, the therapeutic agent comprises a growth factor. In some embodiments, the growth factor is selected from Table 1. In some embodiments, the therapeutic agent comprises a bone morphogenetic protein (BMP). In some embodiments, the therapeutic agent comprises a targeting moiety, and the targeting moiety is non-covalently bound to the three-dimensional structure. In some embodiments, the targeting moiety comprises a polypeptide at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to any one of the sequences in Tables 2-3. In some embodiments, the therapeutic agent comprises a chimeric polypeptide comprising a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to any one of SEQ ID NOs: 433-441.

[0020] Further provided are methods for treating a disease in a subject in need thereof, the method comprising administering a structure or device described herein to the subject. In some embodiments, the disease comprises a bone defect, a cartilage defect, a soft tissue defect, a tendon defect, a fascial defect, a ligament defect, an organ defect, an osteotendinous tissue defect, a skin defect, an osteochondral defect, osteoporosis, avascular necrosis, or a congenital skeletal deformity, or a combination thereof. In some embodiments, the method comprises spinal fusion. In some embodiments, the spinal fusion comprises posterior lumbar fusion (PLF) and / or interbody fusion. In some embodiments, the method comprises bone repair, dental repair, craniomaxillofacial repair, ankle fusion, vertebroplasty, osteoplasty, navicular fracture repair, tendon-bone repair, rib reconstruction, subchondral bone repair, cartilage repair, or surgical implantation of a three-dimensional structure or device, or a combination thereof.

[0021] Further provided is an ink formulation comprising about 55% to about 65% by weight of β-TCP, about 15% to about 25% by weight of PCL, about 5% to about 15% of PEG having a molecular weight of about 500 g / mol to about 15,000 g / mol, and about 5% to about 15% of PEG having a molecular weight of about 25,000 g / mol to about 50,000 g / mol. In some embodiments, the formulation comprises about 60% by weight of β-TCP, about 20% by weight of PCL, about 10% by weight of PEG having a molecular weight of about 500 g / mol to about 15,000 g / mol, and about 10% of PEG having a molecular weight of about 25,000 g / mol to about 50,000 g / mol. In some embodiments, the formulation comprises about 60% by weight β-TCP, about 20% by weight PCL, about 10% by weight PEG having a molecular weight of about 8,000 g / mol, and about 10% by weight PEG having a molecular weight of about 35,000 g / mol. In some embodiments, the formulation comprises about 1% to about 10% of a sacrificial pore-former. In some embodiments, the sacrificial pore-former comprises sucrose.

[0022] Further provided is an ink formulation comprising about 55% to about 65% by weight of β-TCP, about 15% to about 25% by weight of caprolactone / glycolide copolymer, about 5% to about 15% of PEG having a molecular weight of about 500 g / mol to about 15,000 g / mol, and about 5% to about 15% of PEG having a molecular weight of about 25,000 g / mol to about 50,000 g / mol. In some embodiments, the formulation comprises about 60% by weight of β-TCP, about 20% by weight of caprolactone / glycolide copolymer, about 10% by weight of PEG having a molecular weight of about 500 g / mol to about 15,000 g / mol, and about 10% of PEG having a molecular weight of about 25,000 g / mol to about 50,000 g / mol. In some embodiments, the formulation comprises about 60% by weight of β-TCP, about 20% by weight of caprolactone / glycolide copolymer, about 10% by weight of PEG having a molecular weight of about 8,000 g / mol, and about 10% by weight of PEG having a molecular weight of about 35,000 g / mol. In some embodiments, the caprolactone / glycolide copolymer is a caprolactone / glycolide copolymer (95:5). In some embodiments, the caprolactone / glycolide copolymer is a caprolactone / glycolide copolymer (90:10).

[0023] Further provided is a formulation comprising about 55% to about 65% by weight of β-TCP, about 15% to about 25% by weight of poly(D,L-lactide-co-glycolide) copolymer, about 5% to about 15% of PEG having a molecular weight of about 500 g / mol to about 15,000 g / mol, and about 5% to about 15% of PEG having a molecular weight of about 25,000 g / mol to about 50,000 g / mol. In some embodiments, the formulation comprises about 60% by weight of β-TCP, about 20% by weight of poly(D,L-lactide-co-glycolide) copolymer, about 10% by weight of PEG having a molecular weight of about 500 g / mol to about 15,000 g / mol, and about 10% of PEG having a molecular weight of about 25,000 g / mol to about 50,000 g / mol. In some embodiments, the formulation comprises about 60% by weight of β-TCP, about 20% by weight of poly(D,L-lactide-co-glycolide) copolymer, about 10% by weight of PEG having a molecular weight of about 8,000 g / mol, and about 10% by weight of PEG having a molecular weight of about 35,000 g / mol. In some embodiments, the caprolactone / glycolide copolymer is a 50:50 caprolactone / glycolide copolymer.

[0024] Further provided is an ink formulation comprising about 55% to about 65% by weight of β-TCP, about 15% to about 25% by weight of PDS, about 5% to about 15% of PEG having a molecular weight of about 500 g / mol to about 15,000 g / mol, and about 5% to about 15% of PEG having a molecular weight of about 25,000 g / mol to about 50,000 g / mol. In some embodiments, the formulation comprises about 60% by weight of β-TCP, about 20% by weight of PDS, about 10% by weight of PEG having a molecular weight of about 500 g / mol to about 15,000 g / mol, and about 10% of PEG having a molecular weight of about 25,000 g / mol to about 50,000 g / mol. In some embodiments, the formulation comprises about 60% by weight βTCP, about 20% by weight PDS, about 10% by weight PEG having a molecular weight of about 8,000 g / mol, and about 10% PEG having a molecular weight of about 35,000 g / mol.

[0025] Further provided is an ink formulation comprising about 55% to about 65% by weight of β-TCP, about 15% to about 25% by weight of dioxanone / L-lactide copolymer, about 5% to about 15% of PEG having a molecular weight of about 500 g / mol to about 15,000 g / mol, and about 5% to about 15% of PEG having a molecular weight of about 25,000 g / mol to about 50,000 g / mol. In some embodiments, the formulation comprises about 60% by weight of β-TCP, about 20% by weight of dioxanone / L-lactide copolymer, about 10% by weight of PEG having a molecular weight of about 500 g / mol to about 15,000 g / mol, and about 10% of PEG having a molecular weight of about 25,000 g / mol to about 50,000 g / mol. In some embodiments, the formulation comprises about 60% by weight of β-TCP, about 20% by weight of dioxanone / L-lactide copolymer, about 10% by weight of PEG having a molecular weight of about 8,000 g / mol, and about 10% by weight of PEG having a molecular weight of about 35,000 g / mol. In some embodiments, the caprolactone / glycolide copolymer is a 90:10 caprolactone / glycolide copolymer.

[0026] Further provided is an ink formulation comprising about 55% to about 65% by weight of β-TCP, about 15% to about 25% by weight of glycolide / L-lactide copolymer, about 5% to about 15% of PEG having a molecular weight of about 500 g / mol to about 15,000 g / mol, and about 5% to about 15% of PEG having a molecular weight of about 25,000 g / mol to about 50,000 g / mol. In some embodiments, the formulation comprises about 60% by weight of β-TCP, about 20% by weight of glycolide / L-lactide copolymer, about 10% by weight of PEG having a molecular weight of about 500 g / mol to about 15,000 g / mol, and about 10% of PEG having a molecular weight of about 25,000 g / mol to about 50,000 g / mol. In some embodiments, the formulation comprises about 60% by weight β-TCP, about 20% by weight glycolide / L-lactide copolymer, about 10% by weight PEG having a molecular weight of about 8,000 g / mol, and about 10% by weight PEG having a molecular weight of about 35,000 g / mol. In some embodiments, the caprolactone / glycolide copolymer is glycolide / L-lactide copolymer (95:5).

[0027] Further provided is an ink formulation comprising about 55% to about 65% by weight of β-TCP, about 15% to about 25% by weight of poly-l-lactide, about 5% to about 15% of PEG having a molecular weight of about 500 g / mol to about 15,000 g / mol, and about 5% to about 15% of PEG having a molecular weight of about 25,000 g / mol to about 50,000 g / mol. In some embodiments, the formulation comprises about 60% by weight of β-TCP, about 20% by weight of poly-l-lactide, about 10% by weight of PEG having a molecular weight of about 500 g / mol to about 15,000 g / mol, and about 10% of PEG having a molecular weight of about 25,000 g / mol to about 50,000 g / mol. In some embodiments, the formulation comprises about 60% by weight βTCP, about 20% by weight poly-l-lactide, about 10% by weight PEG having a molecular weight of about 8,000 g / mol, and about 10% PEG having a molecular weight of about 35,000 g / mol.

[0028] Further provided is a pellet comprising the formulation.Further provided is a method for preparing a three-dimensional structure, the method comprising using the molded article in a three-dimensional printing process.

[0029] Also provided are three-dimensional structures prepared using the ink formulation. In some embodiments, the structure comprises about 70% to about 80% by weight of β-TCP and about 20% to about 30% by weight of a polymer selected from PCL, PDS, poly-l-lactide, caprolactone / glycolide copolymer, poly(D,L-lactide-co-glycolide) copolymer, dioxanone / L-lactide copolymer, and glycolide / L-lactide copolymer. Also provided are methods for producing three-dimensional structures, the methods comprising depositing the ink formulation in a three-dimensional form, e.g., additive manufacturing. In some embodiments, the methods comprise FFF. In some embodiments, the methods comprise FGF. In some embodiments, the ink formulation comprises the ink formulation described herein. In some embodiments, the ink formulation is in the form of a pellet. In some embodiments, the ink formulation is in the form of a filament.

[0030] Further provided is a method of treating a disease in a subject in need thereof, comprising delivering a structure to an organ or tissue of the subject.

[0031] Further provided is a method of treating a disease in a subject in need thereof, comprising delivering a structure produced by the methods herein to an organ or tissue of the subject.

[0032] In some embodiments, the disease comprises a bone defect, a cartilage defect, a soft tissue defect, a tendon defect, a fascial defect, a ligament defect, an organ defect, an osteotendinous tissue defect, a skin defect, an osteochondral defect, osteoporosis, avascular necrosis, or a congenital skeletal deformity, or a combination thereof. In some embodiments, the method comprises spinal fusion. In some embodiments, the spinal fusion comprises posterior lumbar fusion (PLF) and / or interbody fusion. In some embodiments, the method comprises bone repair, dental restoration, craniomaxillofacial restoration, ankle fusion, vertebroplasty, osteoplasty, navicular fracture repair, tendon-bone repair, rib reconstruction, subchondral bone repair, cartilage repair, or surgical implantation of a three-dimensional structure or device, or a combination thereof. In some embodiments, the method further comprises treating the subject with a therapeutic agent.

[0033] Additionally provided is a method of delivering a therapeutic agent to a subject in need thereof, comprising delivering a device comprising the therapeutic agent and a structure to an organ or tissue of the subject.

[0034] In some embodiments, the therapeutic agent comprises a mammalian growth factor or functional portion thereof. In some embodiments, the therapeutic agent comprises one or more polypeptides, or functional portions thereof, selected from Table 1. In some embodiments, the therapeutic agent comprises a bone morphogenetic protein (BMP). In some embodiments, the therapeutic agent comprises a targeting moiety that non-covalently binds to the structure. In some embodiments, the targeting moiety binds to the printed three-dimensional structure with an affinity of about 1 pM to about 100 μM. In some embodiments, the targeting moiety comprises a polypeptide that is at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to any one of the sequences in Tables 5-6. In some embodiments, the targeting moiety comprises about 2, 3, 4, 5, 6, 7, 8, 9, or 10 sequences selected from the sequences in Tables 5-6. In some embodiments, the therapeutic agent comprises or is a portion of a chimeric polypeptide comprising a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to any one of SEQ ID NOs: 433-441.

[0035] In some embodiments of the devices and / or structures herein, the structures have a density of about 1 g / cm to about 1.5 g / cm. In some embodiments of the devices and / or structures herein, the structures have an open porosity of about 25% to about 40%. In some embodiments of the devices and / or structures herein, the structures have a strut diameter of about 300 μm to about 800 μm.

[0036] The details of one or more embodiments of the disclosure are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the disclosure will be apparent from the description and drawings, and from the claims. [Brief explanation of the drawings]

[0037] [Figure 1A] 1A-1C are images from a scanning electron microscope (SEM image) of an exemplary 3D printed object made with ink formulation #1 as outlined in Example 2. FIG. 1A is an SEM image of the surface of the object at magnification. [Figure 1B] 1A-1B are images from a scanning electron microscope (SEM image) of an exemplary 3D printed object made with ink formulation #1 as outlined in Example 2. FIG. 1B is an SEM image of the side of the object. [Figure 1C] 1A-1C are images from a scanning electron microscope (SEM image) of an exemplary 3D printed object made with ink formulation #1 as outlined in Example 2. FIG. 1C is an SEM image of the surface of the object at a magnified magnification. [Figure 2A] 2A-2C are images from a scanning electron microscope (SEM image) of an exemplary 3D printed object made with ink formulation #2 as outlined in Example 2. Figure 2A is an SEM image of the surface of the object at a magnified magnification. [Figure 2B] 2A and 2B are images from a scanning electron microscope (SEM image) of an exemplary 3D printed object made with ink formulation #2 as outlined in Example 2. FIG. 2B is an SEM image of the side of the object. [Figure 2C] 2A and 2B are images from a scanning electron microscope (SEM image) of an exemplary 3D printed object made with ink formulation #2 as outlined in Example 2. FIG. 2C is an SEM image of the surface of the object at a magnified magnification. [Figure 3A] 1 is an image from a scanning electron microscope (SEM image) of an exemplary 3D printed object made with ink formulation #3 as outlined in Example 2. [Figure 3B] 3A and 3B are images from a scanning electron microscope (SEM image) of an exemplary 3D printed object made with ink formulation #3 as outlined in Example 2. FIG. 3B is an SEM image of the side of the object. [Figure 3C] 3A and 3B are images from a scanning electron microscope (SEM image) of an exemplary 3D printed object made with ink formulation #3 as outlined in Example 2. Figure 3C is an SEM image of the surface of the object at a magnified magnification. [Figure 4A] 4A-4C are images from a scanning electron microscope (SEM image) of an exemplary 3D printed object made with ink formulation #4 as outlined in Example 2. Figure 4A is an SEM image of the surface of the object at a magnified magnification. [Figure 4B]4A and 4B are images from a scanning electron microscope (SEM image) of an exemplary 3D printed object made with ink formulation #4 as outlined in Example 2. FIG. 4B is an SEM image of the side of the object. [Figure 4C] 4A and 4B are images from a scanning electron microscope (SEM image) of an exemplary 3D printed object made with ink formulation #4 as outlined in Example 2. FIG. 4C is an SEM image of the surface of the object at a magnified magnification. [Figure 5A] 5A-5C are images from a scanning electron microscope (SEM image) of an exemplary 3D printed object made with ink formulation #5 as outlined in Example 2. Figure 5A is an SEM image of the surface of the object at magnification. [Figure 5B] 5A and 5B are images from a scanning electron microscope (SEM image) of an exemplary 3D printed object made with ink formulation #5 as outlined in Example 2. FIG. 5B is an SEM image of the side of the object. [Figure 5C] 5A and 5B are images from a scanning electron microscope (SEM image) of an exemplary 3D printed object made with ink formulation #5 as outlined in Example 2. FIG. 5C is an SEM image of the surface of the object at a magnified magnification. [Figure 6A] 6A-6C are images from a scanning electron microscope (SEM image) of an exemplary 3D printed object made with ink formulation #6 as outlined in Example 2. Figure 6A is an SEM image of the surface of the object at magnification. [Figure 6B] 6A and 6B are images from a scanning electron microscope (SEM image) of an exemplary 3D printed object made with ink formulation #6 as outlined in Example 2. Figure 6B is an SEM image of the side of the object. [Figure 6C] 6A-6C are images from a scanning electron microscope (SEM image) of an exemplary 3D printed object made with ink formulation #6 as outlined in Example 2. FIG. 6C is an SEM image of the surface of the object at a magnified magnification. [Figure 7A] 7A-7C are images from a scanning electron microscope (SEM image) of an exemplary 3D printed object made with ink formulation #7 as outlined in Example 2. Figure 7A is an SEM image of the surface of the object at magnification. [Figure 7B]7A and 7B are images from a scanning electron microscope (SEM image) of an exemplary 3D printed object made with ink formulation #7 as outlined in Example 2. Figure 7B is an SEM image of the side of the object. [Figure 7C] 7A-7C are images from a scanning electron microscope (SEM image) of an exemplary 3D printed object made with ink formulation #7 as outlined in Example 2. FIG. 7C is an SEM image of the surface of the object at a magnified magnification. [Figure 8A] 8A-8C are images from a scanning electron microscope (SEM image) of an exemplary 3D printed object made with ink formulation #8 as outlined in Example 2. Figure 8A is an SEM image of the surface of the object at a magnified magnification. [Figure 8B] 8A and 8B are images from a scanning electron microscope (SEM image) of an exemplary 3D printed object made with ink formulation #8 as outlined in Example 2. Figure 8B is an SEM image of the side of the object. [Figure 8C] 8A and 8B are images from a scanning electron microscope (SEM image) of an exemplary 3D printed object made with ink formulation #8 as outlined in Example 2. Figure 8C is an SEM image of the surface of the object at a magnified magnification. [Figure 9A] 9A-9C are images from a scanning electron microscope (SEM image) of an exemplary 3D printed object made with ink formulation #9 as outlined in Example 2. Figure 9A is an SEM image of the surface of the object at a magnified magnification. [Figure 9B] 9A and 9B are images from a scanning electron microscope (SEM image) of an exemplary 3D printed object made with ink formulation #9 as outlined in Example 2. Figure 9B is an SEM image of the side of the object. [Figure 9C] 9A-9C are images from a scanning electron microscope (SEM image) of an exemplary 3D printed object made with ink formulation #9 as outlined in Example 2. FIG. 9C is an SEM image of the surface of the object at a magnified magnification. [Figure 10A] 10A-10C are images from a scanning electron microscope (SEM image) of an exemplary 3D printed object made with ink formulation #10 as outlined in Example 2. Figure 10A is an SEM image of the surface of the object at a magnified magnification. [Figure 10B]10A and 10B are images from a scanning electron microscope (SEM image) of an exemplary 3D printed object made with ink formulation #10 as outlined in Example 2. FIG. 10B is an SEM image of the side of the object. [Figure 10C] 10A-10C are images from a scanning electron microscope (SEM image) of an exemplary 3D printed object made with ink formulation #10 as outlined in Example 2. FIG. 10C is an SEM image of the surface of the object at a magnified magnification. [Figure 11A] 11A-11C are images from a scanning electron microscope (SEM image) of an exemplary 3D printed object made with ink formulation #11 as outlined in Example 2. Figure 11A is an SEM image of the surface of the object at a magnified magnification. [Figure 11B] 11A and 11B are images from a scanning electron microscope (SEM image) of an exemplary 3D printed object made with ink formulation #11 as outlined in Example 2. FIG. 11B is an SEM image of the side of the object. [Figure 11C] 11A-11C are images from a scanning electron microscope (SEM image) of an exemplary 3D printed object made with ink formulation #11 as outlined in Example 2. FIG. 11C is an SEM image of the surface of the object at a magnified magnification. [Figure 12A] 12A and 12B are images from a scanning electron microscope (SEM image) of an exemplary 3D printed object made with ink formulation #12 as outlined in Example 2. Figure 12A is an SEM image of the surface of the object at a magnified magnification. [Figure 12B] 12A and 12B are images from a scanning electron microscope (SEM image) of an exemplary 3D printed object made with ink formulation #12 as outlined in Example 2. Figure 12B is an SEM image of the side of the object. [Figure 12C] 12A and 12B are images from a scanning electron microscope (SEM image) of an exemplary 3D printed object made with ink formulation #12 as outlined in Example 2. Figure 12C is an SEM image of the surface of the object at a magnified magnification. [Figure 13A] FIG. 10 is a photograph of an exemplary flexible three-layer film made with ink formulation #16 3D printed with a 400 micron nozzle as outlined in Example 2. [Figure 13B]1 is an image from a scanning electron microscope (SEM image) of a flexible three-layer membrane at a magnification. [Figure 13C] 1 is an image from a scanning electron microscope (SEM image) of a flexible three-layer membrane at a magnification. [Figure 13D] 1 is an image from a scanning electron microscope (SEM image) of a flexible three-layer membrane at a magnification. [Figure 14A] FIG. 10 is a photograph of an exemplary gyroid scaffold made with ink formulation #16 3D printed with a 400 micron nozzle as outlined in Example 2. [Figure 14B] 1 is an image from a scanning electron microscope (SEM image) of a gyroid scaffold at a magnification. [Figure 14C] 1 is an image from a scanning electron microscope (SEM image) of a gyroid scaffold at a magnification. [Figure 14D] 1 is an image from a scanning electron microscope (SEM image) of a gyroid scaffold at a magnification. [Figure 15A] FIG. 10 is a photograph of an exemplary flexible three-layer film made with ink formulation #18 3D printed with a 400 micron nozzle as outlined in Example 2. [Figure 15B] FIG. 10 is a photograph of a hollow cylinder made with ink formulation #18 3D printed with a 400 micron nozzle as outlined in Example 2. [Figure 16A] FIG. 10 is a photograph of an exemplary flexible three-layer film made with ink formulation #19 3D printed with a 400 micron nozzle as outlined in Example 2. [Figure 16B] FIG. 10 is a photograph of a hollow cylinder made with ink formulation #19 3D printed with a 400 micron nozzle as outlined in Example 2. [Figure 17A] Microscopic images of L929 mouse fibroblast cells after an in vitro cytotoxicity assay performed to measure the cellular response, specifically the toxic effects, upon exposure to extracts from 3D printed scaffolds. [Figure 17B] Cytotoxicity assay results are shown, measured using the following cytotoxicity scale as defined in the ISO 10993-5:2009 standard: DETAILED DESCRIPTION OF THE INVENTION

[0038] Various formulations and structures are provided herein. The structures may be coated with tetherable proteins (e.g., growth factors) for a desired therapeutic effect, such as promoting bone growth after implantation of the tethered structure.

[0039] formulation In one aspect, the present invention provides a formulation for producing a 3D printed structure. As a non-limiting example, the formulation includes a ceramic material, such as calcium phosphate (e.g., tricalcium phosphate, beta tricalcium phosphate, alpha tricalcium phosphate), hydroxyapatite, fluorapatite, bone (e.g., demineralized bone), glass (bioglass), such as silicate, vanadate, and related ceramic minerals, or chelated divalent metal ions, or a combination thereof. In some embodiments, the ceramic material includes beta-tricalcium phosphate (β-TCP). In some embodiments, the formulation is about 30-70, 30-65, 30-60, 30-55, 30-50, 30-45, 30-40, 30-35, 35-70, 35-65, 35-60, 35-55, 35-50, 35-45, 35-40, 40-70, 40-65, 40-60, 40-55, 40-50, 40-45, 45-70, 45-65, 45-60, 45-55, 45-50, 50-70, 50-65, 50-55, 55-70, 55-65, 55-60, 60-70, 60-65, or 65-70 weight percent ceramic of the formulation. For example, the formulation is about 30% by weight, 31% by weight, 32% by weight, 33% by weight, 34% by weight, 35% by weight, 36% by weight, 37% by weight, 38% by weight, 39% by weight, 40% by weight, 41% by weight, 42% by weight, 43% by weight, 44% by weight, 45% by weight, 46% by weight, 47% by weight, 48% by weight, 49% by weight, 50% by weight, 51% by weight, 52% by weight, 53% by weight, 54% by weight, 55% by weight, 56% by weight, 57% by weight, 58% by weight, 59% by weight, 60% by weight, 61% by weight, 62% by weight, 63% by weight, 64% by weight, 65% by weight, 66% by weight, 67% by weight, 68% by weight, 69% by weight, or 70% by weight ceramic. In a non-limiting example, the formulation is about 60% by weight ceramic. In some embodiments, the ceramic is β-TCP. In some embodiments, β-TCP is introduced into the formulation as a powder. In some embodiments, the formulation comprises one or more additional components. Non-limiting examples of additional components include water, polymers (including copolymers), antifoaming agents, dispersants, solvents, particulate or sacrificial pore-forming agents, foaming agents, and plasticizers.

[0040] In some embodiments, the formulation comprises one or more polymers, e.g., about 1, 2, 3, 4, or 5 polymers. Non-limiting examples of polymers include poly(ethylene oxide), poly(propylene oxide), polyethylene glycol (PEG), and polyester. In some embodiments, the polymer is a water-soluble polymer, e.g., PEG. In some embodiments, the formulation comprises about 5-30 weight percent of the polymer in the formulation. In some embodiments, the formulation comprises about 10-30 weight percent of the polymer in the formulation. In some embodiments, the formulation comprises about 20-60 weight percent total polymer. For example, the total polymer comprises two or more polymers in the formulation, and the total percentage of polymer in the formulation is about 20-60 weight percent of the formulation. In one exemplary embodiment, the first polymer is present in about 5-15 weight percent of the formulation, and the second polymer is present in about 5-15 weight percent of the formulation. In some embodiments, the formulation comprises about 30 to about 50 weight percent total polymer. As a non-limiting example, the formulation is about 35-45 weight percent total polymer. In one example, the polymer comprises a poloxamer. Poloxamers are block copolymers of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO). A non-limiting example of a poloxamer is poloxamer 407, such as Pluronic® F-127. In some cases, the formulation comprises about 5-20, 5-15, 5-10, 10-20, 10-15, or 15-20 weight percent poloxamer 407. In another example, the polymer comprises polyethylene glycol (PEG). In some cases, the formulation comprises about 5-30, 5-25, 5-20, 5-15, 5-10, 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30 weight percent PEG. For example, the formulation comprises about 5-30, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 weight percent PEG. In some embodiments, there is a first PEG having a first molecular weight and a second PEG having a second molecular weight.In some embodiments, the PEG can have a molecular weight of 500 g / mol to 35,000 g / mol. In some embodiments, the molecular weight of the PEG is about 500 g / mol, about 1,000 g / mol, about 1,500 g / mol, 20,000 g / mol, about 2,500 g / mol, about 3,000 g / mol, about 3,500 g / mol, about 4,000 g / mol, about 4,500 g / mol, about 5,000 g / mol, about 5,500 g / mol, about 6,000 g / mol, about 6,500 g / mol, about 7,000 g / mol, about 7,500 g / mol, about 8,000 g / mol, about 8,500 g / mol, or about 9,000 g / mol. 00 g / mol, about 9,500 g / mol, about 10,000 g / mol, about 10,500 g / mol, about 11,000 g / mol, about 11,500 g / mol, about 12,000 g / mol, about 12,500 g / mol, about 13,000 g / mol, about 13,500 g / mol, about 14,000 g / mol, about 14,500 g / mol, about 15,000 g / mol, about 15,500 g / mol, about 16,000 g / mol, about 16,500 g / mol, about 17,000 g / mol, about 17,500 g / mol, about 18,000 g / mol, about 18,500 g / mol, about 19,000 g / mol, about 19,500 g / mol, about 20,000 g / mol, about 20,500 g / mol, about 21,000 g / mol, about 21,500 g / mol, about 22,000 g / mol, about 22,500 g / mol, about 23,000 g / mol, about 23,500 g / mol, about 24,000 g / mol, about 24,500 g / mol, about 25,000 g / mol, 25,500 g / mol, 26,000 g / mol , 26,500 g / mol, 27,000 g / mol, 27,500 g / mol, 28,000 g / mol, 28,500 g / mol, 29,000 g / mol, 29,500 g / mol, 30,000 g / mol, 30,500 g / mol, 31,000 g / mol, 31,500 g / mol, 32,000 g / mol, 32,500 g / mol, 33,000 g / mol, 33,500 g / mol, 34,000 g / mol, 34,500 g / mol, or about 35,000 g / mol. In a non-limiting exemplary embodiment, the formulation comprises PEG having a molecular weight of 1,500 g / mol. In a further non-limiting exemplary embodiment, the formulation comprises PEG having a molecular weight of 8,000 g / mol.In a further non-limiting exemplary embodiment, the formulation comprises a PEG having a molecular weight of 20,000 g / mol. In a further non-limiting exemplary embodiment, the formulation comprises a PEG having a molecular weight of 35,000 g / mol. In a non-limiting exemplary embodiment, the formulation comprises about 5-15 weight percent of the first PEG and about 5-15 weight percent of the second PEG. In a non-limiting exemplary embodiment, the formulation comprises about 15 weight percent of the first PEG and about 15 weight percent of the second PEG. In a non-limiting exemplary embodiment, the formulation comprises about 5 weight percent of the first PEG and about 5 weight percent of the second PEG. In a non-limiting exemplary embodiment, the formulation comprises about 10 weight percent of the first PEG and about 10 weight percent of the second PEG. The first PEG may have a lower molecular weight and a lower melt viscosity than the second PEG. For example, the first PEG has a molecular weight of about 500-15,000 g / mol, and the second PEG has a molecular weight of about 25,000-50,000 g / mol. In another example, the polymer includes polydioxanone (PDS). In some cases, the formulation includes about 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30 weight percent PDS. For example, the formulation includes about 15-25, or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 weight percent PDS. In another example, the polymer includes poly-l-lactide. In some cases, the formulation comprises about 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30 weight percent poly-l-lactide. For example, the formulation comprises about 15-25, or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 weight percent poly-l-lactide. As another example, the polymer comprises a polyester. In some embodiments, the polyester comprises a biodegradable polyester such as polycaprolactone (PCL). In some cases, the formulation comprises about 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30 weight percent PCL.For example, the formulation includes about 15-25, or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 weight percent PCL. In some cases, the formulation includes PCL having a molecular weight of 50,000 g / mol. In some embodiments, the polyester includes polyglycolide or poly(glycolic acid) (PGA). In some cases, the formulation may contain about 0.5-20, 0.5-18, 0.5-16, 0.5-14, 0.5-12, 0.5-10, 0.5-8, 0.5-6, 0.5-4, 0.5-2, 1-20, 1-18, 1-16, 1-14, 1-12, 1-10, 1-8, 1-6, 1-4, 1-2, 2-20, 2-18, 2-16, 2-14, 2-12, 2-10, 2-8, 2-6, 2-4 , 3-20, 3-18, 3-16, 3-14, 3-12, 3-10, 3-8, 3-6, 3-4, 4-20, 4-18, 4-16, 4-14, 4-12, 4-10, 4-8, 4-6, 5-20, 5-18, 5-16, 5-14, 5-12, 5-10, 5-8, 5-6, 8-20, 8-18, 8-16, 8-14, 8-12, or 8-10 weight percent PGA. For example, the formulation may contain about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 weight percent PGA. In some cases, the PGA has a molecular weight of about 38,000 to 54,000. In some embodiments, the polyester comprises a polylactide, such as poly(D,L-lactide). In some embodiments, the formulation comprises a hydroxyl group of about 0.5-20, 0.5-18, 0.5-16, 0.5-14, 0.5-12, 0.5-10, 0.5-8, 0.5-6, 0.5-4, 0.5-2, 1-20, 1-18, 1-16, 1-14, 1-12, 1-10, 1-8, 1-6, 1-4, 1-2, 2-20, 2-18, 2-16, 2-14, 2-12, 2-10, 2-8, 2-6, 2-4, and 3 to 20, 3 to 18, 3 to 16, 3 to 14, 3 to 12, 3 to 10, 3 to 8, 3 to 6, 3 to 4, 4 to 20, 4 to 18, 4 to 16, 4 to 14, 4 to 12, 4 to 10, 4 to 8, 4 to 6, 5 to 20, 5 to 18, 5 to 16, 5 to 14, 5 to 12, 5 to 10, 5 to 8, 5 to 6, 8 to 20, 8 to 18, 8 to 16, 8 to 14, 8 to 12, or 8 to 10 weight percent polylactide.For example, the formulation includes about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 weight percent polylactide.

[0041] In some embodiments, the polymer comprises a copolymer. In some embodiments, the copolymer is present in the formulation at about 10-30% by weight. Optionally, the copolymer comprises polyglycolide. Optionally, the copolymer comprises PCL and polyglycolide. For example, the copolymer may comprise about 80-99, 80-98, 80-97, 80-96, 80-95, 80-94, 80-93, 80-92, 80-91, 80-90, 80-89, 80-88, 80-87, 80-86, 80-85, 85-99, 85-98, 85-97, 85-96, 85-95, 85-94, 85-93, 85-92, 85-91, 85-90, 90-99, 90-98, 90-97, 90-96, 90-95, 90-94, 90-93, 90-92, 90-91, 95-99, 95-98, 95-97, or 95-96 mole percent PCL and about 1 and 1-20, 1-18, 1-16, 1-14, 1-12, 1-10, 1-8, 1-6, 1-4, 1-2, 2-20, 2-18, 2-16, 2-14, 2-12, 2-10, 2-8, 2-6, 2-4, 3-20, 3-18, 3-16, 3-14, 3-12, 3-10, 3-8, 3-6, 3-4, 4-20, 4-18, 4-16, 4-14, 4-12, 4-10, 4-8, 4-6, 5-20, 5-18, 5-16, 5-14, 5-12, 5-10, 5-8, 5-6, 8-20, 8-18, 8-16, 8-14, 8-12, or 8-10 mole percent polyglycolide. In some cases, the copolymer comprises about 90-95 mole percent PCL and about 5-10 mole percent polyglycolide. In some cases, the copolymer comprises PDS and polyglycolide.For example, the copolymer may comprise about 80-99, 80-98, 80-97, 80-96, 80-95, 80-94, 80-93, 80-92, 80-91, 80-90, 80-89, 80-88, 80-87, 80-86, 80-85, 85-99, 85-98, 85-97, 85-96, 85-95, 85-94, 85-93, 85-92, 85-91, 85-90, 90-99, 90-98, 90-97, 90-96, 90-95, 90-94, 90-93, 90-92, 90-91, 95-99, 95-98, 95-97, or 95-96 mole percent PDS and about 1 and 1-20, 1-18, 1-16, 1-14, 1-12, 1-10, 1-8, 1-6, 1-4, 1-2, 2-20, 2-18, 2-16, 2-14, 2-12, 2-10, 2-8, 2-6, 2-4, 3-20, 3-18, 3-16, 3-14, 3-12, 3-10, 3-8, 3-6, 3-4, 4-20, 4-18, 4-16, 4-14, 4-12, 4-10, 4-8, 4-6, 5-20, 5-18, 5-16, 5-14, 5-12, 5-10, 5-8, 5-6, 8-20, 8-18, 8-16, 8-14, 8-12, or 8-10 mole percent polyglycolide. In some cases, the copolymer comprises about 90-95 mole percent PDS and about 5-10 mole percent polyglycolide. In some cases, the copolymer comprises a PDS-glycolide copolymer. For example, the formulation comprises about 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, 25-30, or 20 weight percent PDS-glycolide copolymer. In some cases, the copolymer comprises glycolide / L-lactide. In some cases, the formulation comprises about 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30 weight percent glycolide / L-lactide copolymer. For example, the formulation includes about 15 to 25, or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 weight percent glycolide / L-lactide copolymer. In some cases, the copolymer includes glycolide and lactide (e.g., L-lactide).For example, the copolymer may be about 80-99, 80-98, 80-97, 80-96, 80-95, 80-94, 80-93, 80-92, 80-91, 80-90, 80-89, 80-88, 80-87, 80-86, 80-85, 85-99, 85-98, 85-97, 85-96, 85-95 , 85-94, 85-93, 85-92, 85-91, 85-90, 90-99, 90-98, 90-97, 90-96, 90-95, 90-94, 90-93, 90-92, 90-91, 95-99, 95-98, 95-97, or 95-96 mole percent glycolide and about 1 to 20 mole percent glycolide. , 1-18, 1-16, 1-14, 1-12, 1-10, 1-8, 1-6, 1-4, 1-2, 2-20, 2-18, 2-16, 2-14, 2-12, 2-10, 2-8, 2-6, 2-4, 3-20, 3-18, 3-16, 3-14, 3-12, 3-10, 3-8, 3-6, 3-4, 4-20, and 4-18, 4-16, 4-14, 4-12, 4-10, 4-8, 4-6, 5-20, 5-18, 5-16, 5-14, 5-12, 5-10, 5-8, 5-6, 8-20, 8-18, 8-16, 8-14, 8-12, or 8-10 mole percent lactide (e.g., L-lactide). In some cases, the copolymer contains about 85-95 mole percent glycolide and about 5-15 mole percent lactide (e.g., L-lactide), such as a glycolide / L-lactide copolymer (95:5). In some cases, the copolymer includes a caprolactone / glycolide copolymer. In some cases, the formulation comprises about 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30 weight percent caprolactone / glycolide copolymer. For example, the formulation comprises about 15-25, or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 weight percent caprolactone / glycolide copolymer. In some cases, the copolymer comprises caprolactone and glycolide.For example, the copolymer may be a copolymer of about 80-99, 80-98, 80-97, 80-96, 80-95, 80-94, 80-93, 80-92, 80-91, 80-90, 80-89, 80-88, 80-87, 80-86, 80-85, 85-99, 85-98, 85-97, 85-96, 85-95, 85-94, 85-93, 85-92, 85-91, 85-90, 90-99, 90-98, 90-97, 90-96, 90-95, 90-94, 90-93, 90-92, 90-91, 95-99, 95-98, 95-97, or 95-96 mole percent caprolactone. , about 1~20, 1~18, 1~16, 1~14, 1~12, 1~10, 1~8, 1~6, 1~4, 1~2, 2~20, 2~18, 2~16, 2 ~14, 2~12, 2~10, 2~8, 2~6, 2~4, 3~20, 3~18, 3~16, 3~14, 3~12, 3~10, 3~8, 3~6, ​​3 and about 4, 4 to 20, 4 to 18, 4 to 16, 4 to 14, 4 to 12, 4 to 10, 4 to 8, 4 to 6, 5 to 20, 5 to 18, 5 to 16, 5 to 14, 5 to 12, 5 to 10, 5 to 8, 5 to 6, 8 to 20, 8 to 18, 8 to 16, 8 to 14, 8 to 12, or 8 to 10 mole percent glycolide. In some cases, the copolymer comprises about 85 to 95 mole percent caprolactone and about 5 to 15 mole percent glycolide, such as a caprolactone / glycolide copolymer (95:5) or a caprolactone / glycolide copolymer (90:10). In some cases, the copolymer comprises a poly(D,L-lactide-co-glycolide) copolymer. For example, the formulation includes about 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, 25-30, or 20 weight percent poly(D,L-lactide-co-glycolide) copolymer. In some cases, the copolymer includes lactide (e.g., poly(D,L-lactide)) and polyglycolide.For example, the copolymer may be about 35-65, 35-60, 35-55, 35-50, 35-45, 35-40, 40-65, 40-60, 40-55, 40-50, 40-45, 45-65, 45-60, 45-55, 45-50, 50-65, 50-60, 50-55, 55-65, 55-60, 60-65 mole percent lactide (e.g., poly(D,L-lactide)) and about 35-6 5, 35-60, 35-55, 35-50, 35-45, 35-40, 40-65, 40-60, 40-55, 40-50, 40-45, 45-65, 45-60, 45-55, 45-50, 50-65, 50-60, 50-55, 55-65, 55-60, 60-65 mole percent glycolide, such as poly(D,L-lactide-co-glycolide) copolymer (50:50). In some cases, the copolymer comprises lactide (e.g., L-lactide) and PDS. For example, the copolymer may be a copolymer of about 35-65, 35-60, 35-55, 35-50, 35-45, 35-40, 40-65, 40-60, 40-55, 40-50, 40-45, 45-65, 45-60, 45-55, 45-50, 50-65, 50-60, 50-55, 55-65, 55-60, 60-65 mole percent PDS and and about 35-65, 35-60, 35-55, 35-50, 35-45, 35-40, 40-65, 40-60, 40-55, 40-50, 40-45, 45-65, 45-60, 45-55, 45-50, 50-65, 50-60, 50-55, 55-65, 55-60, 60-65 mole percent lactide (L-lactide). In some cases, the copolymer comprises a PDS-L-lactide copolymer. For example, the formulation may contain about 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, 25-30, or 20 weight percent PDS-L-lactide copolymer. In some cases, the copolymer includes dioxanone. In some cases, the copolymer includes dioxanone and lactide (e.g., L-lactide).For example, the copolymer may be about 80-99, 80-98, 80-97, 80-96, 80-95, 80-94, 80-93, 80-92, 80-91, 80-90, 80-89, 80-88, 80-87, 80-86, 80-85, 85-99, 85-98, 85-97, 85-96, 85-95 , 85-94, 85-93, 85-92, 85-91, 85-90, 90-99, 90-98, 90-97, 90-96, 90-95, 90-94, 90-93, 90-92, 90-91, 95-99, 95-98, 95-97, or 95-96 mole percent dioxanone and about 1 to 20 mole percent dioxanone. , 1-18, 1-16, 1-14, 1-12, 1-10, 1-8, 1-6, 1-4, 1-2, 2-20, 2-18, 2-16, 2-14, 2-12, 2-10, 2-8, 2-6, 2-4, 3-20, 3-18, 3-16, 3-14, 3-12, 3-10, 3-8, 3-6, 3-4, 4-20, and 4 to 18, 4 to 16, 4 to 14, 4 to 12, 4 to 10, 4 to 8, 4 to 6, 5 to 20, 5 to 18, 5 to 16, 5 to 14, 5 to 12, 5 to 10, 5 to 8, 5 to 6, 8 to 20, 8 to 18, 8 to 16, 8 to 14, 8 to 12, or 8 to 10 mole percent lactide (e.g., L-lactide). In some cases, the copolymer contains about 85 to 95 mole percent dioxanone and about 5 to 15 mole percent lactide (e.g., L-lactide), such as a dioxanone / L-lactide copolymer (90:10).

[0042] In some cases, copolymers have faster absorption rates than single polymers. For example, the copolymers used in the exemplary embodiments of Ink Formulation #2 (polycaprolactone / polyglycolide copolymer (95:5)), Ink Formulation #3 (polycaprolactone / polyglycolide copolymer (90:10)), and Ink Formulation #4 (poly(D,L-lactide-co-glycolide) copolymer (50:50)) have faster absorption rates than polycaprolactone. Absorption rates vary from slowest to fastest, including poly-l-lactide, polycaprolactone, polycaprolactone / polyglycolide copolymer (95:5), polycaprolactone / glycolide copolymer (90:10), polydioxanone / L-lactide copolymer (90:10), polydioxanone, glycolide / L-lactide copolymer (95:5), and poly(D,L-lactide-co-glycolide) copolymer (50:50).

[0043] In some embodiments, the formulation comprises two or more polymers. In some embodiments, the formulation is about 5-30, 5-25, 5-20, 5-15, 5-10, 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30 weight percent of a first polymer and about 5-30, 5-25, 5-20, 5-15, 5-10, 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30 weight percent of a second polymer. In some embodiments, one polymer is water soluble and the other polymer is not water soluble. For example, the water-soluble polymer is removed from the scaffold after or during fabrication, and the water-insoluble polymer constitutes the structural element of the scaffold. In a non-limiting example, the formulation is about 10-30 weight percent of the first polymer and about 10-30 weight percent of the second polymer, or about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 weight percent of the first polymer and about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 weight percent of the second polymer. In some cases, the first polymer and / or the second polymer comprises PEG. In some cases, the first polymer comprises PCL and the second polymer comprises PEG. In some cases, the first polymer comprises PDS and the second polymer comprises PEG. In some cases, the first polymer comprises poly-l-lactide and the second polymer comprises PEG. In some cases, the first polymer comprises a copolymer and the second polymer comprises PEG. The copolymer can comprise PCL and polyglycolide (e.g., 95 mol% polycaprolactone, 5 mol% polyglycolide; 90 mol% polycaprolactone, 10 mol% polyglycolide). The copolymer can comprise polylactide (e.g., poly(D,L-lactide) and polyglycolide (e.g., 50 mol% poly(D,L-lactide)), 50 mol% polyglycolide, or poly(D,L-lactide-co-glycolide) copolymer (50:50)).The copolymer can include a PDS-glycolide copolymer (e.g., 90 mol% PDS, 10 mol% polyglycolide). The copolymer can include a PDS-L-lactide copolymer-L-lactide copolymer (e.g., 90 mol% PDS, 10 mol% L-lactide or dioxanone / L-lactide copolymer (90:10)). The copolymer can include a glycolide-L-lactide copolymer (e.g., 95 mol% glycolide, 5 mol% L-lactide or glycolide / L-lactide copolymer (95:5)).

[0044] In some embodiments, the formulation comprises one or more microparticles. The microparticles may be pore-forming agents, sometimes referred to as sacrificial pore-forming agents. The microparticles may be water-soluble. The microparticles may comprise salts and / or sugars. Non-limiting examples of microparticles include sodium chloride, calcium chloride, sucrose, trehalose (e.g., α,α trehalose dihydrate), and mannitol (e.g., D-mannitol). Other pore-forming agents include water-soluble polymers such as PEG. In some cases, the microparticles comprise sucrose. In some embodiments, the formulation contains about 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, or 5-6 weight percent of microparticles. In some cases, the formulation contains about 1-10%, or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% microparticles. For example, about 1-10% sucrose. In some embodiments, the microparticles or pore-forming agent have an average size of about 1 micron to about 500 microns in diameter.For example, the diameter may be about 1 micron to about 450 microns, about 1 micron to about 400 microns, about 1 micron to about 350 microns, about 1 micron to about 300 microns, about 1 micron to about 250 microns, about 1 micron to about 200 microns, about 1 micron to about 150 microns, about 50 microns to about 500 microns, about 50 microns to about 450 microns, about 50 microns to about 400 microns, about 50 microns to about 350 microns, about 50 microns to about 300 microns, about 50 microns to about 250 microns, about 50 microns to about 200 microns, about 50 microns to about 150 microns, about 100 microns to about 5 00 microns, about 100 microns to about 450 microns, about 100 microns to about 400 microns, about 100 microns to about 350 microns, about 100 microns to about 300 microns, about 100 microns to about 250 microns, about 100 microns to about 200 microns, about 100 microns to about 150 microns, about 150 microns to about 500 microns, about 150 microns to about 450 microns, about 150 microns to about 400 microns, about 150 microns to about 350 microns, about 150 microns to about 300 microns, about 150 microns to about 250 microns, or about 150 microns to about 200 microns. In some cases, the microparticles or pore-forming agents have an average size of about 50 microns to about 250 microns, about 60 microns to about 240 microns, about 70 microns to about 230 microns, about 80 microns to about 220 microns, or about 90 microns to about 210 microns in diameter. In some embodiments, the microparticles of the pore-forming agent have an average size of about 100 microns to about 200 microns in diameter, e.g., about 110 microns to about 190 microns, about 120 microns to about 180 microns, about 130 microns to about 170 microns, about 140 microns to about 160 microns in diameter, or about 100 microns, about 110 microns, about 120 microns, about 130 microns, about 140 microns, about 150 microns, about 160 microns, about 170 microns, about 180 microns, about 190 microns, or about 200 microns. In some embodiments, the microparticles or pore-forming agent have an average size of about 150 microns in diameter.In some embodiments, upon removal of the particulates or pore-forming agent, the structure formed from the formulation has micropores that provide the structure with additional surface area for contact with the therapeutic agent compared to structures formed with the formulation without the particulates or pore-forming agent. In some embodiments, the micropores of the structure have an average pore size of about 1 micron to about 500 microns, or about 50 microns to about 250 microns, or about 150 microns in diameter.

[0045] In some embodiments, the formulation includes one or more foaming agents. In some embodiments, the foaming agent comprises about 5-20, 5-18, 5-16, 5-14, 5-12, 5-10, 5-8, 5-6, 6-20, 6-18, 6-16, 6-14, 6-12, 6-10, 6-8, 8-20, 8-18, 8-16, 8-14, 8-12, 8-10, 10-20, 10-18, 10-16, 10-14, 10-12, 5-15, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 weight percent foaming agent. In some cases, the foaming agent releases carbon dioxide base during printing to produce a foam structure that can increase the porosity of the 3D printed structure. Non-limiting examples of foaming agents include baking powder (e.g., monocalcium phosphate, sodium bicarbonate, cornstarch) and azodicarbonamide. In some cases, the foaming agent includes sodium bicarbonate. In some cases, the formulation includes about 5 to 15, or 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 weight percent sodium bicarbonate. In some embodiments, the foaming agent provides micropores in the structure having an average diameter of about 1 micron to about 500 microns, or about 50 microns to about 250 microns, or about 150 microns.

[0046] In some embodiments, the formulation comprises a ceramic material (e.g., β-TCP) and a polymer, wherein the polymer comprises PEO, PPO, PDS, PEG, a polyester, a copolymer, or a combination thereof. In some embodiments, the formulation comprises about 30-70, 30-65, 30-60, 30-55, 30-50, 30-45, 30-40, 30-35, 35-70, 35-65, 35-60, 35-55, 35-50, 35-45, 35-40, 40-70, 40-65, 40-60, 40-55, 40-50, 40-45, 45-70, 45-65, 45-60, 45-55, 45-50, 50-70, 50-65, 50-60, 50-55, 55-70, 55-65, 55-60, 60-70, 60-65, or 65-70 weight percent ceramic material (e.g., βTCP), e.g., about 30 weight percent %, 31 wt%, 32 wt%, 33 wt%, 34 wt%, 35 wt%, 36 wt%, 37 wt%, 38 wt%, 39 wt%, 40 wt%, 41 wt%, 42 wt%, 43 wt%, 44 wt%, 45 wt%, 46 wt%, 47 wt%, 48 wt%, 49 wt%, 50 wt%, 51 wt%, 52 wt%, 53 wt%, 54 wt%, 55 wt%, 56 wt%, 57 wt%, 58 wt%, 59 wt%, 60 wt%, 61 wt%, 62 wt%, 63 wt%, 64 wt%, 65 wt%, 66 wt%, 67 wt%, 68 wt%, 69 wt%, or 70 wt% ceramic material (e.g., β-TCP). In some cases, the formulation includes about 5-20, 5-15, 5-10, 10-20, 10-15, or 15-20 weight percent poloxamer 407. In some cases, the formulation includes about 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30 weight percent PEG. In some cases, the formulation includes about 5-15 weight percent first PEG and about 5-15 weight percent second PEG. In some cases, the formulation includes about 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30 weight percent PCL.In some cases, the formulation includes about 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30 weight percent PDS. In some cases, the formulation includes about 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30 weight percent caprolactone / glycolide copolymer (95:5). In some cases, the formulation includes about 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30 weight percent caprolactone / glycolide copolymer (90:10). In some cases, the formulation includes about 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30 weight percent poly(D,L-lactide-co-glycolide) copolymer (50:50). In some cases, the formulation includes about 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30 weight percent dioxanone / L-lactide copolymer (90:10). In some cases, the formulation includes about 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30 weight percent glycolide / L-lactide copolymer (95:5). In some embodiments, the formulation comprises about 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30 weight percent poly-l-lactide. In some embodiments, the formulation further comprises an antifoaming agent. In some embodiments, the formulation further comprises a dispersing agent. In some embodiments, the formulation further comprises a solvent. In some embodiments, the formulation further comprises a plasticizer. In some embodiments, the formulation further comprises a particulate or sacrificial pore former. In some embodiments, the formulation further comprises a foaming agent.

[0047] In some embodiments, the formulation includes a ceramic material (e.g., β-TCP) and a microparticle or sacrificial pore-forming agent. The microparticle may be water-soluble. Non-limiting examples of microparticles include salts and sugars, such as sodium chloride, calcium chloride, sucrose, trehalose (e.g., α,α trehalose dihydrate), and mannitol (e.g., D-mannitol). The pore-forming agent may be a water-soluble polymer, such as PEG. In some embodiments, the formulation comprises about 30-70, 30-65, 30-60, 30-55, 30-50, 30-45, 30-40, 30-35, 35-70, 35-65, 35-60, 35-55, 35-50, 35-45, 35-40, 40-70, 40-65, 40-60, 40-55, 40-50, 40-45, 45-70, 45-65, 45-60, 45-55, 45-50, 50-70, 50-65, 50-60, 50-55, 55-70, 55-65, 55-60, 60-70, 60-65, or 65-70 weight percent ceramic material (e.g., β-TCP), e.g., about 30 weight percent %, 31 wt%, 32 wt%, 33 wt%, 34 wt%, 35 wt%, 36 wt%, 37 wt%, 38 wt%, 39 wt%, 40 wt%, 41 wt%, 42 wt%, 43 wt%, 44 wt%, 45 wt%, 46 wt%, 47 wt%, 48 wt%, 49 wt%, 50 wt%, 51 wt%, 52 wt%, 53 wt%, 54 wt%, 55 wt%, 56 wt%, 57 wt%, 58 wt%, 59 wt%, 60 wt%, 61 wt%, 62 wt%, 63 wt%, 64 wt%, 65 wt%, 66 wt%, 67 wt%, 68 wt%, 69 wt%, or 70 wt% ceramic material (e.g., β-TCP). In some embodiments, the formulation comprises about 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, or 5-6 weight percent of microparticles. In some embodiments, the microparticles comprise sucrose. In some embodiments, the formulation comprises about 10-30 weight percent or about 5-15 weight percent of a sacrificial pore-forming agent, such as a polymer. In some embodiments, the formulation further comprises water.In some embodiments, the formulation further comprises a polymer. In some embodiments, the formulation further comprises an antifoaming agent. In some embodiments, the formulation further comprises a dispersing agent. In some embodiments, the formulation further comprises a solvent. In some embodiments, the formulation further comprises a plasticizer. In some embodiments, the formulation further comprises a foaming agent.

[0048] In some embodiments, the formulation comprises a ceramic material (e.g., β-TCP) and a foaming agent. Non-limiting examples of foaming agents include baking powder (e.g., monocalcium phosphate, sodium bicarbonate, cornstarch) and azodicarbonamide. The foaming agent may comprise sodium bicarbonate. In some embodiments, the formulation comprises about 30-70, 30-65, 30-60, 30-55, 30-50, 30-45, 30-40, 30-35, 35-70, 35-65, 35-60, 35-55, 35-50, 35-45, 35-40, 40-70, 40-65, 40-60, 40-55, 40-50, 40-45, 45-70, 45-65, 45-60, 45-55, 45-50, 50-70, 50-65, 50-60, 50-55, 55-70, 55-65, 55-60, 60-70, 60-65, or 65-70 weight percent ceramic material (e.g., β-TCP), e.g., about 30 weight percent %, 31 wt%, 32 wt%, 33 wt%, 34 wt%, 35 wt%, 36 wt%, 37 wt%, 38 wt%, 39 wt%, 40 wt%, 41 wt%, 42 wt%, 43 wt%, 44 wt%, 45 wt%, 46 wt%, 47 wt%, 48 wt%, 49 wt%, 50 wt%, 51 wt%, 52 wt%, 53 wt%, 54 wt%, 55 wt%, 56 wt%, 57 wt%, 58 wt%, 59 wt%, 60 wt%, 61 wt%, 62 wt%, 63 wt%, 64 wt%, 65 wt%, 66 wt%, 67 wt%, 68 wt%, 69 wt%, or 70 wt% ceramic material (e.g., β-TCP). In some embodiments, the effervescent agent comprises about 5-20, 5-18, 5-16, 5-15, 5-14, 5-12, 5-10, 5-8, 5-6, 6-20, 6-18, 6-16, 6-14, 6-12, 6-10, 6-8, 8-20, 8-18, 8-16, 8-14, 8-12, 8-10, 10-20, 10-18, 10-16, 10-14, 10-12, 5-15, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 weight percent effervescent agent. In some embodiments, the formulation comprises about 5-15, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 weight percent sodium bicarbonate. In some embodiments, the formulation further comprises water. In some embodiments, the formulation further comprises a polymer.In some embodiments, the formulation further comprises an antifoaming agent. In some embodiments, the formulation further comprises a dispersing agent. In some embodiments, the formulation further comprises a solvent. In some embodiments, the formulation further comprises a plasticizer. In some embodiments, the formulation further comprises a particulate or a sacrificial pore former.

[0049] In another aspect, the formulation includes a ceramic material and one or more polymers. In some embodiments, the formulation includes about 30% to about 70% ceramic material (e.g., β-TCP). For example, the formulation may include about 30-70, 30-65, 30-60, 30-55, 30-50, 30-45, 30-40, 30-35, 35-70, 35-65, 35-60, 35-55, 35-50, 35-45, 35-40, 40-70, 40-65, 40-60, 40-55, 40-50, 40-45, 45-70, 45-65, 45-60, 45-55, 45-50, 50-70, 50-65, 50-6 In some embodiments, the formulation comprises a first polymer, e.g., about 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30 weight percent of the ceramic material (e.g., β-TCP). The first polymer can be polycaprolactone (PCL). The first polymer can be polydioxanone (PDS). The first polymer can be poly-l-lactide. The first polymer can be a copolymer, such as caprolactone / glycolide copolymer, poly(D,L-lactide-co-glycolide) copolymer, dioxanone / L-lactide copolymer, or glycolide / L-lactide copolymer. In some embodiments, the formulation includes a second polymer, such as about 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30 weight percent of the second polymer. The first polymer and / or the second polymer can be water-soluble or water-insoluble. In some embodiments, the formulation includes a first polymer, a second polymer, and / or a third polymer. The third polymer can be water soluble or water insoluble. The second polymer can be polyethylene glycol (PEG).The second polymer can be PEG with a MW of about 8000 g / mol. The third polymer can be PEG. The third polymer can be PEG with a MW of about 35,000 g / mol. In a non-limiting embodiment, the formulation comprises about 30-70 wt% ceramic, about 10-30 wt% first polymer, and about 10-30 wt% second polymer. In a non-limiting embodiment, the formulation comprises about 30-70 wt% ceramic, about 10-30 wt% first polymer, about 5-15 wt% second polymer, and about 5-15 wt% third polymer. For example, the formulation can comprise about 30-70 wt% β-TCP, about 10-30 wt% PCL, and about 10-30 wt% PEG. As another example, the formulation may contain about 30-70% by weight of β-TCP, about 10-30% by weight of PCL, about 5-15% by weight of PEG (8000 MW), and about 5-15% by weight of PEG (35000 MW). As another example, the formulation may contain about 30-70% by weight of β-TCP, about 10-30% by weight of PDS, and about 10-30% by weight of PEG. As another example, the formulation may contain about 30-70% by weight of β-TCP, about 10-30% by weight of PDS, about 5-15% by weight of PEG (8000 MW), and about 5-15% by weight of PEG (35000 MW). As another example, the formulation may contain about 30-70% by weight of β-TCP, about 10-30% by weight of poly-l-lactide, and about 10-30% by weight of PEG. As another example, a formulation may contain about 30-70% by weight of β-TCP, about 10-30% by weight of poly-l-lactide, about 5-15% by weight of PEG (8000 MW), and about 5-15% by weight of PEG (35000 MW). As another example, a formulation may contain about 30-70% by weight of β-TCP, about 10-30% by weight of copolymer, and about 10-30% by weight of PEG. As another example, a formulation may contain about 30-70% by weight of β-TCP, about 10-30% by weight of copolymer, and about 5-15% by weight of PEG (8000 MW), and about 5-15% by weight of PEG (35000 MW). Non-limiting exemplary copolymers include caprolactone / glycolide copolymer, poly(D,L-lactide-co-glycolide) copolymer, dioxanone / L-lactide copolymer, or glycolide / L-lactide copolymer.

[0050] In some further embodiments, the formulation comprises microparticles and / or a pore-forming agent. The microparticles may be water-soluble. In some cases, the microparticles comprise sucrose. In some embodiments, the formulation comprises about 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, or 5-6 weight percent microparticles. For example, the formulation may comprise about 30-70 weight percent βTCP, about 10-30 weight percent PCL, about 10-30 weight percent PEG, and about 1-10 weight percent microparticles. In some embodiments, the formulation may contain about 30-70% by weight of β-TCP, about 10-30% by weight of PCL, PDS, poly-l-lactide, caprolactone / glycolide copolymer, poly(D,L-lactide-co-glycolide) copolymer, dioxanone / L-lactide copolymer, or glycolide / L-lactide copolymer, about 5-15% by weight of 8000 MW PEG, about 5-15% by weight of 35,000 MW PEG, and about 1-10% by weight of microparticles. In some embodiments, the formulation contains PEG, where PEG is a pore-forming agent. In some embodiments, PEG is present at about 10-30% by weight, or at about 5-15% by weight of 8000 MW PEG and about 5-15% by weight of 35,000 MW PEG.

[0051] In some further embodiments, the formulation comprises an effervescent agent. In some cases, the effervescent agent comprises sodium bicarbonate. In some embodiments, the formulation comprises about 5-20, 5-18, 5-16, 5-14, 5-12, 5-10, 5-8, 5-6, 6-20, 6-18, 6-16, 6-14, 6-12, 6-10, 6-8, 8-20, 8-18, 8-16, 8-14, 8-12, 8-10, 10-20, 10-18, 10-16, 10-14, 10-12, 5-15, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 weight percent effervescent agent. For example, a formulation may contain about 30-70% by weight of β-TCP, about 10-30% by weight of PCL, about 10-30% by weight of PEG, and about 5-20% by weight of a foaming agent. For example, a formulation may contain about 30-70% by weight of β-TCP, about 10-30% by weight of PCL, PDS, poly-l-lactide, caprolactone / glycolide copolymer, poly(D,L-lactide-co-glycolide) copolymer, dioxanone / L-lactide copolymer, or glycolide / L-lactide copolymer, about 5-15% by weight of 8000 MW PEG, about 5-15% by weight of 35,000 MW PEG, and about 5-20% by weight of a foaming agent.

[0052] In some further embodiments, the polymer of the formulation is a copolymer, such as a copolymer of PCL and polyglycolide. For example, the copolymer may comprise about 80-99, 80-98, 80-97, 80-96, 80-95, 80-94, 80-93, 80-92, 80-91, 80-90, 80-89, 80-88, 80-87, 80-86, 80-85, 85-99, 85-98, 85-97, 85-96, 85-95, 85-94, 85-93, 85-92, 85-91, 85-90, 90-99, 90-98, 90-97, 90-96, 90-95, 90-94, 90-93, 90-92, 90-91, 95-99, 95-98, 95-97, or 95-96 mole percent PCL and about 1 and 1-20, 1-18, 1-16, 1-14, 1-12, 1-10, 1-8, 1-6, 1-4, 1-2, 2-20, 2-18, 2-16, 2-14, 2-12, 2-10, 2-8, 2-6, 2-4, 3-20, 3-18, 3-16, 3-14, 3-12, 3-10, 3-8, 3-6, 3-4, 4-20, 4-18, 4-16, 4-14, 4-12, 4-10, 4-8, 4-6, 5-20, 5-18, 5-16, 5-14, 5-12, 5-10, 5-8, 5-6, 8-20, 8-18, 8-16, 8-14, 8-12, or 8-10 mole percent polyglycolide. In some cases, the copolymer comprises about 90-95 mol percent PCL and about 5-10 mol percent polyglycolide. In one example, the formulation may comprise about 30-70 wt% β-TCP, about 10-30 wt% copolymer of PCL and polyglycolide (e.g., 95 mol% polycaprolactone, 5 mol% polyglycolide), and about 10-30 wt% PEG. In one example, the formulation may comprise about 30-70 wt% β-TCP, about 10-30 wt% copolymer of PCL and polyglycolide (e.g., 90 mol% polycaprolactone, 10 mol% polyglycolide), and about 10-30 wt% PEG.In one example, the formulation may contain about 30-70% by weight β-TCP, about 10-30% by weight of a copolymer of PCL and polyglycolide (e.g., 90 mol% polycaprolactone, 10 mol% polyglycolide, which may be referred to as caprolactone / glycolide copolymer (90:10)), and about 5-15% by weight of 8000 MW PEG and about 5-15% by weight of 35,000 MW PEG. In one example, the formulation may contain about 30-70% by weight β-TCP, about 10-30% by weight of a copolymer of PCL and polyglycolide (e.g., 95 mol% polycaprolactone, 5 mol% polyglycolide), and about 5-15% by weight of 8000 MW PEG and about 5-15% by weight of 35,000 MW PEG.

[0053] In some further embodiments, the polymer of the formulation is a copolymer, such as a copolymer of PDS and polyglycolide. For example, the copolymer may comprise about 80-99, 80-98, 80-97, 80-96, 80-95, 80-94, 80-93, 80-92, 80-91, 80-90, 80-89, 80-88, 80-87, 80-86, 80-85, 85-99, 85-98, 85-97, 85-96, 85-95, 85-94, 85-93, 85-92, 85-91, 85-90, 90-99, 90-98, 90-97, 90-96, 90-95, 90-94, 90-93, 90-92, 90-91, 95-99, 95-98, 95-97, or 95-96 mole percent PDS and about 1 and 1-20, 1-18, 1-16, 1-14, 1-12, 1-10, 1-8, 1-6, 1-4, 1-2, 2-20, 2-18, 2-16, 2-14, 2-12, 2-10, 2-8, 2-6, 2-4, 3-20, 3-18, 3-16, 3-14, 3-12, 3-10, 3-8, 3-6, 3-4, 4-20, 4-18, 4-16, 4-14, 4-12, 4-10, 4-8, 4-6, 5-20, 5-18, 5-16, 5-14, 5-12, 5-10, 5-8, 5-6, 8-20, 8-18, 8-16, 8-14, 8-12, or 8-10 mole percent polyglycolide. In some cases, the copolymer comprises about 90-95 mol percent PDS and about 5-10 mol percent polyglycolide. In one example, the formulation may comprise about 30-70 wt% β-TCP, about 10-30 wt% copolymer of PDS and polyglycolide (e.g., 90 mol% PDS, 10 mol% polyglycolide), and about 10-30 wt% PEG. In one example, the formulation may comprise about 30-70 wt% β-TCP, about 10-30 wt% copolymer of PDS and polyglycolide (e.g., 90 mol% PDS, 10 mol% polyglycolide), and about 5-15 wt% 8000 MW PEG and about 5-15 wt% 35,000 MW PEG.

[0054] In some further embodiments, the polymer of the formulation is a copolymer, such as a copolymer of poly(D,L-lactide) and glycolide. For example, the copolymer may be about 30-50, 31-49, 32-48, 33-47, 34-46, 35-45, 36-44, 37-43, 38-42, 39-41, 80-99, 80-98, 80-97, 80-96, 80-95, 80-94, 80-93, 80-92, 80-91, 80-90, 80-89, 80-88, 80-87, 80-86, 80-87 5, 85-99, 85-98, 85-97, 85-96, 85-95, 85-94, 85-93, 85-92, 85-91, 85-90, 90-99, 90-98, 90-97, 90-96, 90-95, 90-94, 90-93, 90-92, 90-91, 95-99, 95-98, 95-97, or 95-96 mole percent poly(DL-lactide) ) and about 30-50, 31-49, 32-48, 33-47, 34-46, 35-45, 36-44, 37-43, 38-42, 39-41, 1-20, 1-18, 1-16, 1-14, 1-12, 1-10, 1-8, 1-6, 1-4, 1-2, 2-20, 2-18, 2-16, 2-14, 2-12, 2-10, 2-8, 2-6, 2-4, 3-20, 3-1 In some cases, the copolymer comprises about 50 mole percent poly(DL-lactide) and about 50 mole percent glycolide. In one example, the formulation may include about 30-70% by weight β-TCP, about 10-30% by weight copolymer of poly(DL-lactide) and glycolide (e.g., 50 mol% poly(DL-lactide), 50 mol% glycolide), and about 10-30% by weight PEG.In one example, the formulation may include about 30-70% by weight β-TCP, about 10-30% by weight copolymer of poly(DL-lactide) and glycolide (e.g., 50 mol% poly(DL-lactide), 50 mol% polyglycolide), and about 5-15% by weight 8000 MW PEG, about 5-15% by weight 35,000 MW PEG.

[0055] In some further embodiments, the polymer of the formulation is a copolymer, such as a copolymer of PDS and lactide. For example, the copolymer may comprise about 80-99, 80-98, 80-97, 80-96, 80-95, 80-94, 80-93, 80-92, 80-91, 80-90, 80-89, 80-88, 80-87, 80-86, 80-85, 85-99, 85-98, 85-97, 85-96, 85-95, 85-94, 85-93, 85-92, 85-91, 85-90, 90-99, 90-98, 90-97, 90-96, 90-95, 90-94, 90-93, 90-92, 90-91, 95-99, 95-98, 95-97, or 95-96 mole percent PDS. Approximately 1~20, 1~18, 1~16, 1~14, 1~12, 1~10, 1~8, 1~6, 1~4, 1~2, 2~20, 2~18, 2~16, 2~ 14, 2~12, 2~10, 2~8, 2~6, 2~4, 3~20, 3~18, 3~16, 3~14, 3~12, 3~10, 3~8, 3~6, ​​3 and about 4, 4-20, 4-18, 4-16, 4-14, 4-12, 4-10, 4-8, 4-6, 5-20, 5-18, 5-16, 5-14, 5-12, 5-10, 5-8, 5-6, 8-20, 8-18, 8-16, 8-14, 8-12, or 8-10 mole percent lactide. In some cases, the copolymer comprises about 90-95 mole percent PDS and about 5-10 mole percent lactide. In one example, the formulation may contain about 30-70 weight percent β-TCP, about 10-30 weight percent PDS and lactide copolymer (e.g., 90 mole percent PDS, 10 mole percent lactide), and about 10-30 weight percent PEG. In one example, the formulation may include about 30-70% by weight β-TCP, about 10-30% by weight copolymer of PDS and lactide (e.g., 90 mol% PDS, 10 mol% lactide), and about 5-15% by weight 8000 MW PEG, about 5-15% by weight 35,000 MW PEG.

[0056] In some further embodiments, the polymer of the formulation is a copolymer, such as a copolymer of glycolide and lactide. For example, the copolymer may be a copolymer of about 80-99, 80-98, 80-97, 80-96, 80-95, 80-94, 80-93, 80-92, 80-91, 80-90, 80-89, 80-88, 80-87, 80-86, 80-85, 85-99, 85-98, 85-97, 85-96, 85-95, 85-94, 85-93, 85-92, 85-91, 85-90, 90-99, 90-98, 90-97, 90-96, 90-95, 90-94, 90-93, 90-92, 90-91, 95-99, 95-98, 95-97, or 95-96 mole percent glycolide. , about 1~20, 1~18, 1~16, 1~14, 1~12, 1~10, 1~8, 1~6, 1~4, 1~2, 2~20, 2~18, 2~16, 2 ~14, 2~12, 2~10, 2~8, 2~6, 2~4, 3~20, 3~18, 3~16, 3~14, 3~12, 3~10, 3~8, 3~6, The copolymer may comprise 3-4, 4-20, 4-18, 4-16, 4-14, 4-12, 4-10, 4-8, 4-6, 5-20, 5-18, 5-16, 5-14, 5-12, 5-10, 5-8, 5-6, 8-20, 8-18, 8-16, 8-14, 8-12, or 8-10 mole percent lactide. In some cases, the copolymer comprises about 90-95 mole percent glycolide and about 5-10 mole percent lactide. In one example, the formulation may comprise about 30-70% by weight β-TCP, about 10-30% by weight copolymer of glycolide and lactide (e.g., 95 mole% glycolide, 10 mole% lactide), and about 10-30% by weight PEG. In one example, the formulation may include about 30-70% by weight β-TCP, about 10-30% by weight glycolide and lactide copolymer (e.g., 95 mol% glycolide, 5 mol% lactide), and about 5-15% by weight 8000 MW PEG, about 5-15% by weight 35,000 MW PEG.

[0057] In other aspects, the formulation includes a ceramic material and one or more polymers. In some embodiments, the formulation includes about 30% to about 70% ceramic material (e.g., β-TCP). For example, the formulation may include about 30-70, 30-65, 30-60, 30-55, 30-50, 30-45, 30-40, 30-35, 35-70, 35-65, 35-60, 35-55, 35-50, 35-45, 35-40, 40-70, 40-65, 40-60, 40-55, 40-50, 40-45, 45-70, 45-65, 45-60, 45-55, 45-50, 50-70, 50-65, 50-6 In some embodiments, the formulation comprises a first polymer, e.g., about 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30 weight percent of the ceramic material (e.g., β-TCP). The first polymer can be a copolymer. In some embodiments, the copolymer comprises lactide (e.g., poly(D,L-lactide)) and polyglycolide. In some embodiments, the copolymer comprises a poly(D,L-lactide-co-glycolide) copolymer. In some embodiments, the copolymer comprises about 50 mol% poly(D,L-lactide) and about 50 mol% polyglycolide. In some embodiments, the copolymer comprises a caprolactone / glycolide (e.g., 90:10, 95:5) copolymer, a poly(D,L-lactide-co-glycolide) (e.g., 50:50) copolymer, a dioxanone / L-lactide (e.g., 90:10) copolymer, or a glycolide / L-lactide (e.g., 95:5) copolymer. In some embodiments, the formulation comprises a second polymer, for example, about 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30 weight percent of the second polymer.The second polymer can be polyethylene glycol (PEG). In non-limiting embodiments, the formulation comprises about 30-70% by weight of the ceramic, about 10-30% by weight of the first polymer, and about 10-30% by weight of the second polymer. For example, the formulation can comprise about 30-70% by weight of β-TCP, about 10-30% by weight of the copolymer, and about 10-30% by weight of PEG. In some embodiments, the formulation can comprise about 30-70% by weight of β-TCP, about 10-30% by weight of the copolymer, about 5-15% by weight of 8000 MW PEG, and about 5-15% by weight of 35,000 MW PEG.

[0058] In some further embodiments, the formulation comprises a ceramic material and one or more polymers. In some embodiments, the formulation comprises about 30% to about 70% ceramic material (e.g., β-TCP). For example, the formulation may comprise about 30-70, 30-65, 30-60, 30-55, 30-50, 30-45, 30-40, 30-35, 35-70, 35-65, 35-60, 35-55, 35-50, 35-45, 35-40, 40-70, 40-65, 40-60, 40-55, 40-50, 40-45, 45-70, 45-65, 45-60, 45-55, 45-50, 50-70, 50-65, 50-6 In some embodiments, the formulation comprises a first polymer, e.g., about 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30 weight percent of the ceramic material (e.g., β-TCP). The first polymer may be a copolymer, such as a copolymer of dioxanone and lactide (eg, L-lactide).For example, the copolymer may be a copolymer of about 80-99, 80-98, 80-97, 80-96, 80-95, 80-94, 80-93, 80-92, 80-91, 80-90, 80-89, 80-88, 80-87, 80-86, 80-85, 85-99, 85-98, 85-97, 85-96, 85-95, 85-94, 85-93, 85-92, 85-91, 85-90, 90-99, 90-98, 90-97, 90-96, 90-95, 90-94, 90-93, 90-92, 90-91, 95-99, 95-98, 95-97, or 95-96 mole percent dioxanone and , about 1~20, 1~18, 1~16, 1~14, 1~12, 1~10, 1~8, 1~6, 1~4, 1~2, 2~20, 2~18, 2~16, 2 ~14, 2~12, 2~10, 2~8, 2~6, 2~4, 3~20, 3~18, 3~16, 3~14, 3~12, 3~10, 3~8, 3~6, and 3-4, 4-20, 4-18, 4-16, 4-14, 4-12, 4-10, 4-8, 4-6, 5-20, 5-18, 5-16, 5-14, 5-12, 5-10, 5-8, 5-6, 8-20, 8-18, 8-16, 8-14, 8-12, or 8-10 mole percent lactide. In some embodiments, the copolymer comprises about 90-95 mole percent dioxanone and about 5-10 mole percent lactide. In some embodiments, the formulation includes a second polymer, for example, about 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30 weight percent of the second polymer. The second polymer can be polyethylene glycol (PEG). In a non-limiting embodiment, the formulation comprises about 30-70% by weight of the ceramic, about 10-30% by weight of the first polymer, and about 10-30% by weight of the second polymer. For example, the formulation can comprise about 30-70% by weight of β-TCP, about 10-30% by weight of the copolymer, and about 10-30% by weight of PEG. In one example, the formulation can comprise about 30-70% by weight of β-TCP, about 10-30% by weight of a copolymer of PDS and lactide (e.g., 90 mol% dioxanone, 10 mol% L-lactide), and about 10-30% by weight of PEG.In some embodiments, the formulation may include about 30-70% by weight β-TCP, about 10-30% by weight copolymer of PDS and lactide (e.g., 90 mol% dioxanone, 10 mol% L-lactide), and about 5-15% by weight 8000 MW PEG, about 5-15% by weight 35,000 MW PEG.

[0059] In one aspect, the formulation has a low viscosity, which may be useful during manufacturing for extrusion through a small-diameter nozzle. The nozzle may have a diameter of about 240 μm to about 500 μm, or about 280 μm to about 450 μm, or about 240 μm to about 850 μm, e.g., about 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 700, 720, 740, 760, 780, 800, 820, 840, or 850 μm. In a non-limiting exemplary embodiment, the formulation is melt-mixed in a double asymmetric centrifugal mixer to produce a homogeneous liquid ink. A mixture of low- and high-viscosity PEGs can be used to minimize oozing of molten ink from the nozzle of a 3D printer during non-printing operations, but the viscosity of the molten ink can be adjusted to allow flow through nozzles with diameters of several hundred microns when the screw extruder is activated for printing operations. Higher molecular weight PEGs are generally harder and stronger than lower molecular weight PEGs, so their incorporation also improves the mechanical strength and hardness of the source material.

[0060] In one aspect, the formulation has a high viscosity that can be useful during manufacturing by forming the formulation into filaments, which may then be used in hot fused filament manufacturing.

[0061] In one aspect, the formulation is in the form of a filament. For example, as further described in Example 2, the ink formulation was prepared into a filament for use in 3D printing structures on a fused filament fabrication (FFF) 3D printer. In some embodiments, the filament formulation has a diameter of about 1 to about 3 mm, or about 1 to about 2.75 mm, about 1 to about 2.5 mm, about 1 to about 2.25 mm, about 1 to about 2 mm, about 1 to about 1.75 mm, about 1 to about 1.5 mm, about 1.25 to about 3 mm, about 1.25 to about 2.75 mm, about 1.25 to about 2.5 mm, about 1.25 to about 2.25 mm, about 1.25 to about 2 mm, about 1.25 to about 1.75 mm, or about 1.25 to about 1.5 mm. mm, about 1.5 to about 3 mm, about 1.5 to about 2.75 mm, about 1.5 to about 2.5 mm, about 1.5 to about 2.25 mm, about 1.5 to about 2 mm, about 1.5 to about 1.75 mm, about 1.75 to about 3 mm, about 1.75 to about 2.75 mm, about 1.75 to about 2.5 mm, about 1.75 to about 2.25 mm, about 1.75 to about 2 mm, about 2 to about 3 mm, about 2 to about 2.75 mm, about 2 to about 2.5 mm, or about 2 to about 2.25 mm. As a non-limiting example, the filament preparation has a diameter of about 1.5 mm to about 2 mm, or about 1.5 mm, about 1.75 mm, or about 2 mm.

[0062] In one embodiment, the formulation is in the form of pellets. For example, as further described in Example 2, the ink formulation was prepared into pellets for use in 3D structures. In some embodiments, the pellets can be processed into filaments. In some embodiments, the pellets can be processed into powders. In some embodiments, the pellets have a length of about 1 to about 6 mm, or about 1 to about 5.5 mm, about 1 to about 5 mm, about 1 to about 4.5 mm, about 1 to about 4 mm, about 1 to about 3.5 mm, about 1 to about 3 mm, about 1 to about 2.5 mm, about 1 to about 2 mm, about 1 to about 1.5 mm, about 1.5 to about 6 mm, about 1.5 to about 5.5 mm, about 1.5 to about 5 mm, about 1.5 to about 4.5 mm, about 1.5 to about 4 mm, about 1.5 to about 3.5 mm, about 1.5 to about 3 mm, about 1.5 to about 2.5 mm, about 2 to about 6 mm, about 2 to about 5.5 mm, about 2 to about 5 mm, about 2 to about 4.5 mm, about 2 to about 4 mm, about 2 to about 3.5 mm, about 2 to about 3 mm, about 2 to about 2.5 mm, or about 2.5 to about 6 mm. mm, about 2.5 to about 5.5 mm, about 2.5 to about 5 mm, about 2.5 to about 4.5 mm, about 2.5 to about 4 mm, about 2.5 to about 3.5 mm, about 2.5 to about 3 mm, about 3 to about 6 mm, about 3 to about 5.5 mm, about 3 to about 5 mm, about 3 to about 4.5 mm, about 3 to about 4 mm, about 3 to about 3.5 mm, about 3.5 to about 6 mm, about 3.5 to about 5.5 mm, about 3.5 to about 5 mm, about 3.5 to about 4.5 mm, about 3.5 to about 4 mm, about 4 to about 6 mm, about 4 to about 5.5 mm, about 4 to about 5 mm, about 4 to about 4.5 mm, about 4.5 to about 6 mm, about 4.5 to about 5.5 mm, about 4.5 to about 5 mm, about 5 to about 6 mm, about 5 to about 5.5 mm, or about 5.5 to about 6 mm. As non-limiting exemplary embodiments, the pellets have a length of about 2.5 mm to about 4.5 mm, or about 2.5 mm, about 3 mm, about 3.5 mm, about 4 mm, or about 4.5 mm. In some embodiments, the pellets encompass a variety of different shapes, including spears, rods, granules, blocks, particles, and particles of any suitable shape.

[0063] In one aspect, the formulation is in the form of a powder. In some embodiments, the powder can be produced from pellets. In a non-limiting exemplary embodiment, the components of the formulation are melt mixed into a homogeneous ink and freeze-milled to form a powder. In a non-limiting exemplary embodiment, the components of the formulation are dissolved in a solvent-based slurry and spray-dried to form a powder. In some embodiments, the powder is used for selective laser sintering.

[0064] 3D printed structure In another aspect, the present invention provides a 3D printed structure.The structure can be prepared using the formulation and / or manufacturing method described herein.As used herein, structure includes scaffold, and vice versa.

[0065] In some embodiments, the three-dimensional structure has micropores. The micropores may be formed after removing particulates or pore-forming agents. The micropores may be formed by using a foaming agent in the formulation. In some embodiments, the micropores provide the structure with additional surface area for contact with a therapeutic agent compared to a structure without micropores. In a non-limiting example, the therapeutic agent comprises a targeting moiety that non-covalently binds to the ceramic material of the structure.

[0066] In some embodiments, the micropores have an average diameter of about 1 micron to about 500 microns. For example, the diameter may be about 1 micron to about 450 microns, about 1 micron to about 400 microns, about 1 micron to about 350 microns, about 1 micron to about 300 microns, about 1 micron to about 250 microns, about 1 micron to about 200 microns, about 1 micron to about 150 microns, about 50 microns to about 500 microns, about 50 microns to about 450 microns, about 50 microns to about 400 microns, about 50 microns to about 350 microns, about 50 microns to about 300 microns, about 50 microns to about 250 microns, about 50 microns to about 200 microns, about 50 microns to about 150 microns, or about 100 microns to about 5 Micropores may have an average diameter of about 100 microns to about 450 microns, about 100 microns to about 400 microns, about 100 microns to about 350 microns, about 100 microns to about 300 microns, about 100 microns to about 250 microns, about 100 microns to about 200 microns, about 100 microns to about 150 microns, about 150 microns to about 500 microns, about 150 microns to about 450 microns, about 150 microns to about 400 microns, about 150 microns to about 350 microns, about 150 microns to about 300 microns, about 150 microns to about 250 microns, or about 150 microns to about 200 microns. In some cases, the micropores have an average diameter of about 50 microns to about 250 microns, about 60 microns to about 240 microns, about 70 microns to about 230 microns, about 80 microns to about 220 microns, or about 90 microns to about 210 microns. In some embodiments, the micropores have an average diameter of about 100 microns to about 200 microns, e.g., a diameter of about 110 microns to about 190 microns, about 120 microns to about 180 microns, about 130 microns to about 170 microns, about 140 microns to about 160 microns, or about 100 microns, about 110 microns, about 120 microns, about 130 microns, about 140 microns, about 150 microns, about 160 microns, about 170 microns, about 180 microns, about 190 microns, or about 200 microns. In some embodiments, the micropores have an average diameter of about 150 microns.

[0067] In some embodiments, the micropores have an average diameter of about 1 micron to about 50 microns, e.g., about 1 micron to about 45 microns, about 1 micron to about 40 microns, about 1 micron to about 35 microns, about 1 micron to about 30 microns, about 1 micron to about 25 microns, about 1 micron to about 20 microns, about 1 micron to about 15 microns, about 1 micron to about 10 microns, about 10 microns to about 50 microns, about 10 microns to about 45 microns, about 10 microns to about 40 microns, about 10 microns to about 35 microns, about 10 microns to about 30 microns, about 10 microns to about 25 ... The average particle size is about 10 microns to about 20 microns, about 10 microns to about 15 microns, about 20 microns to about 50 microns, about 20 microns to about 45 microns, about 20 microns to about 40 microns, about 20 microns to about 35 microns, about 20 microns to about 30 microns, about 20 microns to about 25 microns, about 30 microns to about 50 microns, about 30 microns to about 45 microns, about 30 microns to about 40 microns, about 30 microns to about 35 microns, about 40 microns to about 50 microns, or about 40 microns to about 45 microns.

[0068] In an exemplary embodiment, the microporosity of the scaffold results in a hydrophilic scaffold, ie, liquids readily wick throughout the scaffold via capillary forces from the interconnected microporosity.

[0069] In some embodiments, the three-dimensional structure has a density of about 1 g / cm 3 ~About 3g / cm 3 In some embodiments, the three-dimensional structure has a density of about 1 g / cm 3 ~Approx. 2g / cm 3 (e.g., about 1, 1.1, 1.15, 1.2, 1.25, 1.3, 1.35, 1.4, 1.45, 1.5, 1.55, 1.6, 1.65, 1.7, 1.75, 1.8, 1.85, 1.9, 1.95, or 2 g / cm 3 or any value in between).

[0070] In some embodiments, the three-dimensional structure has an open porosity of about 15% to about 50%, about 15% to about 45%, about 15% to about 40%, about 20% to about 50%, about 20% to about 45%, about 25% to about 40%, about 25% to about 50%, about 25% to about 45%, or about 25% to about 40%. In some embodiments, the open porosity is about 25% to about 40%, e.g., about 25%, 30%, 35%, or 40%, or any value therebetween.

[0071] In some embodiments, the three-dimensional structure has a strut diameter of about 300 μm to about 600 μm, about 325 μm to about 600 μm, about 350 μm to about 600 μm, about 375 μm to about 600 μm, about 400 μm to about 600 μm, about 425 μm to about 600 μm, about 450 μm to about 600 μm, about 475 μm to about 600 μm, about 500 μm to about 600 μm, about 525 μm to about 600 μm, about 550 μm to about 600 μm, about 300 μm to about 575 μm, about 325 μm to about 575 μm, about 350 μm to about 575 μm, about 375 μm to about 575 μm, about 400 μm to about 575 μm. μm, approximately 425 μm to approximately 575 μm, approximately 450 μm to approximately 575 μm, approximately 475 μm to approximately 575 μm, approximately 500 μm to approximately 575 μm, approximately 52 5μm to approx. 575μm, approx. 550μm to approx. 575μm, approx. 300μm to approx. 550μm, approx. 325μm to approx. 550μm, approx. 350μm to approx. 5 50μm, approximately 375μm to approximately 550μm, approximately 400μm to approximately 550μm, approximately 425μm to approximately 550μm, approximately 450μm to approximately 550μm, approximately 475μm~about 550μm, about 500μm~about 550μm, about 525μm~about 550μm, about 300μm~about 525μm, about 325μm~about 525μm, about 350μm to about 525μm, about 375μm to about 525μm, about 400μm to about 525μm, about 425μm to about 525μm, Approximately 450μm to approximately 525μm, approximately 475μm to approximately 525μm, approximately 500μm to approximately 525μm, approximately 300μm to approximately 500μm, approximately 325μm ~Approx. 500μm, approx. 350μm ~ approx. 500μm, approx. 375μm ~ approx. 500μm, approx. 400μm ~ approx. 500μm, approx. 425μm ~ approx. 500μm m, approximately 450 μm to approximately 500 μm, approximately 475 μm to approximately 500 μm, approximately 300 μm to approximately 475 μm, approximately 325 μm to approximately 475 μm, approximately 350 μm ~ approx. 475 μm, approx. 375 μm ~ approx. 475 μm, approx. 400 μm ~ approx. 475 μm, approx. 425 μm ~ approx. 475 μm, approx. 450 μm ~ approx. 47 5μm, about 300μm to about 450μm, about 325μm to about 450μm, about 350μm to about 450μm, about 375μm to about 450μm, about 4 00 μm to about 450 μm, about 425 μm to about 450 μm, about 300 μm to about 400 μm, about 325 μm to about 400 μm, about 350 μm to about 400 μm, or about 375 μm to about 400 μm, about 300 to about 850, about 325 to about 850, about 350 to about 850, about 375 to about 850,About 400 to about 850, about 425 to about 850, about 450 to about 850, about 475 to about 850, about 500 to about 850, about 525 to about 850, about 550 to about 850, about 575 to about 850, about 600 to about 850, about 625 to about 850, about 650 to about 850, about 675 to about 850, about 700 to about 850 about 850, about 725 to about 850, about 750 to about 850, about 775 to about 850, about 800 to about 850, about 825 to about 850, about 300 to about 825, about 325 to about 825, about 350 to about 825, about 375 to about 825, about 400 to about 825, about 425 to about 825, about 450 to about 825, About 475 to about 825, about 500 to about 825, about 525 to about 825, about 550 to about 825, about 575 to about 825, about 600 to about 825, about 625 to about 825, about 650 to about 825, about 675 to about 825, about 700 to about 825, about 725 to about 825, about 750 to about 825, about 775 to About 825, about 800 to about 825, about 825 to about 825, about 300 to about 800, about 325 to about 800, about 350 to about 800, about 375 to about 800, about 400 to about 800, about 425 to about 800, about 450 to about 800, about 475 to about 800, about 500 to about 800, about 525 to about 800, About 550 to about 800, about 575 to about 800, about 600 to about 800, about 625 to about 800, about 650 to about 800, about 675 to about 800, about 700 to about 800, about 725 to about 800, about 750 to about 800, about 775 to about 800, about 300 to about 775, about 325 to about 775, about 350 to about 775, about 375 to about 775, about 400 to about 775, about 425 to about 775, about 450 to about 775, about 475 to about 775, about 500 to about 775, about 525 to about 775, about 550 to about 775, about 575 to about 775, about 600 to about 775, about 625 to about 775, about 650 to about 775, About 675 to about 775, about 700 to about 775, about 725 to about 775, about 750 to about 775, about 300 to about 750, about 325 to about 750, about 350 to about 750, about 375 to about 750, about 400 to about 750, about 425 to about 750, about 450 to about 750, about 475 to about 750, about 500 to about 750 about 750, about 525 to about 750, about 550 to about 750, about 575 to about 750, about 600 to about 750, about 625 to about 750, about 650 to about 750, about 675 to about 750, about 700 to about 750, about 725 to about 750, about 300 to about 725, about 325 to about 725, about 350 to about 725,About 375 to about 725, about 400 to about 725, about 425 to about 725, about 450 to about 725, about 475 to about 725, about 500 to about 725, about 525 to about 725, about 550 to about 725, about 575 to about 725, about 600 to about 725, about 625 to about 725, about 650 to about 725, about 675 to about 725, about 700 to about 725, about 300 to about 700, about 325 to about 700, about 350 to about 700, about 375 to about 700, about 4 00 to about 700, about 425 to about 700, about 450 to about 700, about 475 to about 700, about 500 to about 700, about 525 to about 700, about 550 to about 700, about 575 to about 700, about 600 to about 700, about 625 to about 700, about 650 to about 700, about 675 to about 700, about 300 to about 675, about 325 to about 675, about 350 to about 675, about 375 to about 675, about 400 to about 675, about 425 to about 675, about 450 to about 675, about 475 to about 675, about 500 to about 675, about 525 to about 675, about 550 to about 675, about 575 to about 675, about 600 to about 675, about 625 to about 675, about 650 to about 675, about 300 to about 650, about 325 to about 650, about 350 to about 650, about 375 to about 650, about 400 to about 650, about 425 to about 650, about 450 to about 650, about 475 to about 650, about 500 to about 650, about 525 to about 650, about 550 to about 650, about 575 to about 650, about 600 to about 650, about 625 to about 650, about 300 to about 625, about 325 to about 625, about 350 to about 625, about 375 to about 625, about 400 to about 625, about 425 to about 625, about 450 to about 625, about 475 to about 625, about 500 to about 625, about 525 to about 625, about 550 to about 625, about 575 to about 625, or about 600 to about 625 μm.

[0072] In some embodiments, the structure comprises a ceramic material such as calcium phosphate. In some embodiments, the structure comprises a ceramic material having a thickness of about 50-100, 50-95, 50-90, 50-85, 50-80, 50-75, 50-70, 50-65, 50-60, 50-55, 55-100, 55-95, 55-90, 55-85, 55-80, 55-75, 55-70, 55-65, 55-60, 60~100, 60~95, 60~90, 60~85, 60~80, 60~75, 60~70, 60~65, 65~100, 65~95, 65~90, 65~85, 65~80, 65~75, 65~70, 70~100, 70~95, 70~90, 70~85, 70~80, 70~75, 75~ 100, 75-95, 75-90, 75-85, 75-80, 80-100, 80-95, 80-90, 80-85, 85-100, 85-95, 85-90, 90-100, 90-95, 95-100, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 6 96, 97, 98, 99, or 100 percent ceramic material. In some cases, the ceramic material is a calcium phosphate, such as β-tricalcium phosphate (β-TCP).

[0073] In a non-limiting example, the structure has about 50-90% ceramic material, such as β-TCP. In some cases, the structure has about 50, 55, 60, 65, 70, 75, 80, 85, or 90% ceramic material, such as β-TCP. In some embodiments, the structure has about 10-50% polymer, such as polycaprolactone (PCL) or polydioxanone (PDS), or poly-l-lactide. In some cases, the structure has about 10, 15, 20, 25, 30, 35, 40, 45, or 50% polymer, such as PCL or PDS or poly-l-lactide. Exemplary structures include about 85-90% by weight ceramic (e.g., β-TCP) and about 10-15% by weight polymer (e.g., PCL or PDS or poly-l-lactide), about 80-85% by weight ceramic (e.g., β-TCP) and about 15-20% by weight polymer (e.g., PCL or PDS or poly-l-lactide), about 75-80% by weight ceramic (e.g., β-TCP) and about 20-25% by weight polymer (e.g., PCL or PDS or poly-l-lactide), about 70-75% by weight ceramic (e.g., β-TCP) and about 25-30% by weight polymer (e.g., PCL or PDS or poly-l-lactide), and about 65-70% by weight ceramic (e.g., β-TCP) and about 30-35% by weight polymer (e.g., PCL or PDS or poly-l-lactide). ), about 60-65 wt% ceramic (e.g., β-TCP) and about 35-40 wt% polymer (e.g., PCL or PDS or poly-l-lactide), about 55-60 wt% ceramic (e.g., β-TCP) and about 40-45 wt% polymer (e.g., PCL or PDS or poly-l-lactide), about 50-55 wt% ceramic (e.g., β-TCP) and about 45-50 wt% polymer (e.g., PCL or PDS or poly-l-lactide), about 90 wt% ceramic (e.g., β-TCP) and about 10 wt% polymer (e.g., PCL or PDS or poly-l-lactide), about 89 wt% ceramic (e.g., β-TCP) and about 11 wt% polymer (e.g., PCL or PDS or poly-l-lactide), about 88 wt% ceramic (e.g.,β-TCP) and about 12% by weight of a polymer (e.g., PCL or PDS or poly-l-lactide), about 87% by weight of a ceramic (e.g., β-TCP) and about 13% by weight of a polymer (e.g., PCL or PDS or poly-l-lactide), about 86% by weight of a ceramic (e.g., β-TCP) and about 14% by weight of a polymer (e.g., PCL or PDS or poly-l-lactide), about 85% by weight of a ceramic (e.g., β-TCP) and about 15% by weight of a polymer (e.g., PCL or PDS or poly-l-lactide), about 84% by weight about 83 wt% ceramic (e.g., β-TCP) and about 17 wt% polymer (e.g., PCL or PDS or poly-l-lactide); about 82 wt% ceramic (e.g., β-TCP) and about 18 wt% polymer (e.g., PCL or PDS or poly-l-lactide); about 81 wt% ceramic (e.g., β-TCP) and about 19 wt% polymer (e.g., PCL or PDS or poly-l-lactide). about 80% by weight of ceramic (e.g., β-TCP) and about 20% by weight of polymer (e.g., PCL or PDS or poly-l-lactide); about 79% by weight of ceramic (e.g., β-TCP) and about 21% by weight of polymer (e.g., PCL or PDS or poly-l-lactide); about 78% by weight of ceramic (e.g., β-TCP) and about 22% by weight of polymer (e.g., PCL or PDS or poly-l-lactide); about 77% by weight of ceramic (e.g., β-TCP) and about 23% by weight of polymer (e.g., PCL or PDS or poly-l-lactide), about 76 wt% ceramic (e.g., β-TCP) and about 24 wt% polymer (e.g., PCL or PDS or poly-l-lactide), about 75 wt% ceramic (e.g., β-TCP) and about 25 wt% polymer (e.g., PCL or PDS or poly-l-lactide), about 74 wt% ceramic (e.g., β-TCP) and about 26 wt% polymer (e.g., PCL or PDS or poly-l-lactide), about 73 wt% ceramic (e.g., β-TCP) and about 27 wt% polymer (e.g.,PCL or PDS or poly-l-lactide), about 72 wt% ceramic (e.g., β-TCP) and about 28 wt% polymer (e.g., PCL or PDS or poly-l-lactide), about 71 wt% ceramic (e.g., β-TCP) and about 29 wt% polymer (e.g., PCL or PDS or poly-l-lactide), about 70 wt% ceramic (e.g., β-TCP) and about 30 wt% polymer (e.g., PCL or PDS or poly-l-lactide), about 69 wt% ceramic (e.g., β-TCP) and about 31 wt% polymer % polymer (e.g., PCL or PDS or poly-l-lactide), about 68 wt. % ceramic (e.g., β-TCP) and about 32 wt. % polymer (e.g., PCL or PDS or poly-l-lactide), about 67 wt. % ceramic (e.g., β-TCP) and about 33 wt. % polymer (e.g., PCL or PDS or poly-l-lactide), about 66 wt. % ceramic (e.g., β-TCP) and about 34 wt. % polymer (e.g., PCL or PDS or poly-l-lactide), about 65 wt. % ceramic (e.g., β-TCP) and about 34 wt. % polymer (e.g., PCL or PDS or poly-l-lactide). P) and about 35 wt% polymer (e.g., PCL or PDS or poly-l-lactide), about 64 wt% ceramic (e.g., β-TCP) and about 36 wt% polymer (e.g., PCL or PDS or poly-l-lactide), about 63 wt% ceramic (e.g., β-TCP) and about 37 wt% polymer (e.g., PCL or PDS or poly-l-lactide), about 62 wt% ceramic (e.g., β-TCP) and about 38 wt% polymer (e.g., PCL or PDS or poly-l-lactide), about 61 wt% ceramic ceramic (e.g., β-TCP) and about 39% by weight of a polymer (e.g., PCL or PDS or poly-l-lactide); about 60% by weight of a ceramic (e.g., β-TCP) and about 40% by weight of a polymer (e.g., PCL or PDS or poly-l-lactide); about 59% by weight of a ceramic (e.g., β-TCP) and about 41% by weight of a polymer (e.g., PCL or PDS or poly-l-lactide); about 58% by weight of a ceramic (e.g., β-TCP) and about 42% by weight of a polymer (e.g., PCL or PDS or poly-l-lactide);About 57% by weight of ceramic (e.g., β-TCP) and about 43% by weight of polymer (e.g., PCL or PDS or poly-l-lactide), about 56% by weight of ceramic (e.g., β-TCP) and about 44% by weight of polymer (e.g., PCL or PDS or poly-l-lactide), about 55% by weight of ceramic (e.g., β-TCP) and about 45% by weight of polymer (e.g., PCL or PDS or poly-l-lactide), about 54% by weight of ceramic (e.g., β-TCP) and about 46% by weight of polymer (e.g., PCL or PDS or poly-l-lactide), % ceramic (e.g., β-TCP) and about 47% polymer (e.g., PCL or PDS or poly-l-lactide), about 52% ceramic (e.g., β-TCP) and about 48% polymer (e.g., PCL or PDS or poly-l-lactide), about 51% ceramic (e.g., β-TCP) and about 49% polymer (e.g., PCL or PDS or poly-l-lactide), about 50% ceramic (e.g., β-TCP) and about 50% polymer (e.g., PCL or PDS or poly-l-lactide),

[0074] The structure can be fabricated using 3D printing from an ink containing about 30-70 wt% β-TCP powder, about 10-30 wt% of a first polymer, and about 10-30 wt% of a second polymer. In some cases, the structure can be fabricated using 3D printing from an ink containing about 30-70 wt% β-TCP powder, about 10-30 wt% of a first polymer, about 5-15 wt% of a second polymer, and about 5-15 wt% of a third polymer. In some cases, the structure can be fabricated using 3D printing from an ink containing about 30-70 wt% β-TCP powder, about 10-30 wt% of a first polymer, about 5-15 wt% of a second polymer (8000 MW PEG), and about 5-15 wt% of a third polymer (35,000 MW PEG). In some cases, the first polymer includes PCL. In some cases, the first polymer includes PDS. In some cases, the first polymer includes poly-l-lactide. In some cases, the second polymer includes PEG. In some cases, the third polymer includes PEG. In some cases, the ink further includes about 1-10% by weight of particulates (e.g., sucrose). In some cases, the ink further includes about 5-20% by weight of a foaming agent (e.g., sodium bicarbonate).

[0075] In some embodiments, the three-dimensional structure has a density of about 1 g / cm 3 ~Approx. 2g / cm 3 , or about 1 g / cm 3 ~Approx. 1.5g / cm 3 In some embodiments, the three-dimensional structure has an open porosity of about 20% to about 40%, about 25% to about 35%, e.g., about 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35%. In some embodiments, the three-dimensional structure has a strut diameter of about 300 μm to about 900 μm, about 300 μm to about 400 μm, or about 500 μm to about 900 μm.

[0076] In some embodiments, the three-dimensional structure has a density of about 1 g / cm 3 ~Approx. 2g / cm 3 , or approximately 1.25 g / cm 3~Approx. 1.75g / cm 3 In some embodiments, the three-dimensional structure has an open porosity of about 20% to about 40%, about 25% to about 35%, e.g., about 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35%. In some embodiments, the three-dimensional structure has a strut diameter of about 400 μm to about 500 μm, about 400 μm to about 450 μm, or about 425 μm to about 450 μm.

[0077] In some embodiments, the three-dimensional structure has a density of about 1 g / cm 3 ~Approx. 2g / cm 3 , or about 1 g / cm 3 ~Approx. 1.5g / cm 3 In some embodiments, the three-dimensional structure has an open porosity of about 30% to about 50%, about 35% to about 45%, e.g., about 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, or 45%. In some embodiments, the three-dimensional structure has a strut diameter of about 325 μm to about 425 μm, about 350 μm to about 400 μm, or about 360 μm to about 390 μm.

[0078] In some embodiments, the three-dimensional structure has a density of about 1 g / cm 3 ~Approx. 2g / cm 3 , or about 1 g / cm 3 ~Approx. 1.5g / cm 3 In some embodiments, the three-dimensional structure has an open porosity of about 30% to about 50%, about 35% to about 45%, e.g., about 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, or 45%. In some embodiments, the three-dimensional structure has a strut diameter of about 350 μm to about 450 μm, about 350 μm to about 400 μm, or about 380 μm to about 405 μm.

[0079] In a non-limiting example, the structure comprises about 50-90% ceramic material, such as β-TCP. In some cases, the structure comprises about 50, 55, 60, 65, 70, 75, 80, 85, or 90% ceramic material, such as β-TCP. In some embodiments, the structure comprises about 10-50% copolymer, such as polycaprolactone / polyglycolide copolymer (PCL / PGA, e.g., 90:10, 95:5), poly(D,L-lactide-co-glycolide) copolymer (PLGA, e.g., 50:50), PDS-glycolide copolymer (PDS / PGA, e.g., 90:10), PDS-L-lactide copolymer (PDS / PLA, e.g., 90:10), or dioxanone / L-lactide copolymer (e.g., 90:10). In some cases, the structure has about 10, 15, 20, 25, 30, 35, 40, 45, or 50% polymer, such as PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide). Exemplary structures include about 85-90% by weight ceramic (e.g., β-TCP) and about 10-15% by weight polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or dioxanone / L-lactide), about 80-85% by weight ceramic (e.g., β-TCP) and about 15-20% by weight polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, dioxanone / L-lactide). / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)), about 75-80 wt% ceramic (e.g., β-TCP) and about 20-25 wt% polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)).L-lactide-co-glycolide), about 70-75 wt % ceramic (e.g., β-TCP) and about 25-30 wt % polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)), about 65-70 wt % ceramic (e.g., β-TCP) and about 30-35 wt % polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)). about 60-65 wt % ceramic (e.g., β-TCP) and about 35-40 wt % polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)), about 55-60 wt % ceramic (e.g., β-TCP) and about 40-45 wt % polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)), about 50-55 wt% ceramic (e.g., β-TCP) and about 45-50 wt% polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)), about 90 wt% ceramic ceramic (e.g., β-TCP) and about 10 wt. % polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)), about 89 wt. % ceramic (e.g., β-TCP) and about 11 wt. % polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)).L-lactide-co-glycolide), about 88% by weight ceramic (e.g., β-TCP) and about 12% by weight polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)), about 87% by weight ceramic (e.g., β-TCP) and about 13% by weight polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)), caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)), about 86% by weight ceramic (e.g., β-TCP) and about 14% by weight polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)), about 85% by weight ceramic (e.g., β-TCP) and about 15% by weight polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)), about 84 wt % ceramic (e.g., β-TCP) and about 16 wt % polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)), about 83 wt % ceramic (e.g., , β-TCP) and about 17 wt. % polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)), about 82 wt. % ceramic (e.g., β-TCP) and about 18 wt. % polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)).L-lactide-co-glycolide), about 81 wt % ceramic (e.g., β-TCP) and about 19 wt % polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)), about 80 wt % ceramic (e.g., β-TCP) and about 20 wt % polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)). prolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)), about 79% by weight ceramic (e.g., β-TCP) and about 21% by weight polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)), about 78% by weight ceramic (e.g., β-TCP) and about 22% by weight polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)). DS / PGA, PDS / PLA, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)), about 77% by weight ceramic (e.g., β-TCP) and about 23% by weight polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)), about 76% by weight ceramic (e.g., β-TCP). , β-TCP) and about 24 wt. % polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)), about 75 wt. % ceramic (e.g., β-TCP) and about 25 wt. % polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)).L-lactide-co-glycolide), about 74% by weight ceramic (e.g., β-TCP) and about 26% by weight polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)), about 73% by weight ceramic (e.g., β-TCP) and about 27% by weight polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)). prolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)), about 72 wt % ceramic (e.g., β-TCP) and about 28 wt % polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)), about 71 wt % ceramic (e.g., β-TCP) and about 29 wt % polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)). S / PGA, PDS / PLA, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)), about 70% by weight ceramic (e.g., β-TCP) and about 30% by weight polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)), about 69% by weight ceramic (e.g., β-TCP) and about 31% by weight of a polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)), about 68% by weight of a ceramic (e.g., β-TCP) and about 32% by weight of a polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)).L-lactide-co-glycolide)), about 67 wt % ceramic (e.g., β-TCP) and about 33 wt % polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)), about 66 wt % ceramic (e.g., β-TCP) and about 34 wt % polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)), about 65 wt % ceramic (e.g., β-TCP) and about 35 wt % polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLGA, A, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)), about 64 wt% ceramic (e.g., β-TCP) and about 36 wt% polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)), about 63 wt% ceramic ceramic (e.g., β-TCP) and about 37% by weight of polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)), about 62% by weight of ceramic (e.g., β-TCP) and about 38% by weight of polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)). / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)), about 61% by weight ceramic (e.g., β-TCP) and about 39% by weight polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)), about 60% by weight ceramic (e.g., β-TCP) and about 40% by weight polymer - (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)), about 59 wt. % ceramic (e.g., β-TCP) and about 41 wt. % polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)).L-lactide-co-glycolide), about 58% by weight ceramic (e.g., β-TCP) and about 42% by weight polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)), about 57% by weight ceramic (e.g., β-TCP) and about 43% by weight polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)). caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)), about 56% by weight ceramic (e.g., β-TCP) and about 44% by weight polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)), about 55% by weight ceramic (e.g., β-TCP) and about 45% by weight polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)), about 54 wt % ceramic (e.g., β-TCP) and about 46 wt % polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)), about 53 wt % ceramic (e.g., , β-TCP) and about 47% by weight of polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)), about 52% by weight of ceramic (e.g., β-TCP) and about 48% by weight of polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)).L-lactide-co-glycolide), about 51% by weight ceramic (e.g., β-TCP) and about 49% by weight polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)), about 50% by weight ceramic (e.g., β-TCP) and about 50% by weight polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, dioxanone / L-lactide, caprolactone / glycolide, glycolide / L-lactide, or poly(D,L-lactide-co-glycolide)).

[0080] In some embodiments, the composition of the ink formulations herein is modified to optimize the specific surface area. The surface area may be optimized for combination with a particular therapeutic agent. For example, the structure may be configured to have a specific surface area of ​​approximately 0.2 to 2 m for combination with a BMP protein (e.g., tBMP-2). 2 In some embodiments, the surface area of ​​the structures herein is about 0.2 to 2, 0.2 to 1.8, 0.2 to 1.6, 0.2 to 1.4, 0.2 to 1.2, 0.2 to 1, 0.2 to 0.8, 0.2 to 0.6, 0.2 to 0.4, 0.4 to 2, 0.4 to 1.8, 0.4 to 1.6, 0.4 to 1.4, 0.4 to 1.2, 0.4 to 1, 0.4 to 0.8, 0.4 to 0.6, 0.6 to 2, 0.6 to 1.8, 0.6 to 1.6, 0.6-1.4, 0.6-1.2, 0.6-1, 0.6-0.8, 0.8-2, 0.8-1.8, 0.8-1.6, 0.8-1.4, 0.8-1.2, 0.8-1, 1-2, 1-1.8, 1-1.6, 1-1.4, 1-1.2, 1.2-2, 1.2-1.8, 1.2-1.6, 1.2-1.4, 1.4-2, 1.4-1.8, 1.4-1.6, 1.6-2, 1.6-1.8, or 1.8-2 m 2 / g. In some embodiments, the surface area is calculated by Brunauer-Emmett-Teller (BET) by gas physisorption.

[0081] In some embodiments, the composition of the ink formulations herein is modified to optimize the absorption rate of one or more materials of the scaffold. For example, polymers are selected based on their absorption rate, ranging from slowest to fastest, such as polycaprolactone, polycaprolactone / polyglycolide copolymer (95:5), polycaprolactone / glycolide copolymer (90:10), polydioxanone / L-lactide copolymer (90:10), and poly(D,L-lactide-co-glycolide) copolymer (50:50).

[0082] Manufacturing method In another aspect, a method for manufacturing a structure using 3D printing technology is provided.

[0083] In some embodiments, the method involves syringe-based melt extrusion bioprinting. Exemplary inks in this method may have low viscosity to allow the ink to be extruded through a narrow nozzle. Non-limiting examples of manufacturing methods using this method are described in Example 2, for example, for printing ink formulations #1, #2, #3, and #4.

[0084] In one embodiment, the method is an extrusion method, including 3D printing, in which material is extruded from a nozzle.

[0085] In some embodiments, extrusion methods include bioprinting (syringe-based pneumatic printing) or fused granulation (FGF), in which pellets of raw material are fed from a hopper into a mini-screw extrusion head, which melts the material and extrudes it through a fine nozzle. In some embodiments, the ink is formed into small (e.g., 2-5 mm) pellets or granules. In an exemplary embodiment, the ink comprises a plurality of pellets or granules having an average diameter of X, where at least 90% of the plurality have an individual diameter of X ± 0.5 mm. For example, the plurality of pellets or granules has an average diameter of 2 mm, and at least 90% of the plurality have an individual diameter of 1.5-2.5 mm. As another example, the plurality of pellets or granules has an average diameter of 5 mm, and at least 90% of the plurality have an individual diameter of 4.5-5.5 mm.

[0086] In some embodiments, the method includes fused filament fabrication (FFF). Exemplary inks in this method can be formed into filaments for printing on FFF 3D printers. Non-limiting examples of manufacturing methods using this method are described in Example 2, for example, for printing ink formulations #5 and #6.

[0087] In some embodiments, the method includes pelletized fused deposition modeling. Fused deposition modeling (FDM) is an additive manufacturing process. Three-dimensional objects are formed by the extrusion and deposition of individual layers of thermoplastic material. FDM involves the molten extrusion of a filamentary material through a heated nozzle and deposition as a thin, solid layer on a platform. The thermoplastic polymer material is fed into a temperature-controlled FDM extrusion head and heated to a semi-liquid state. The FDM extrusion head then extrudes the material in ultra-thin layers, depositing it onto a base with high precision. The material solidifies and builds on the previous layer. In this way, multiple parts are manufactured in layers, with each layer being built by extruding small beads of material, called "loads," in a specific pattern so that the layer is covered by adjacent loads. After each layer is completed, the height of the extrusion head is raised, and subsequent layers are built to form the multiple parts. FDM is typically used to manufacture solid models. To manufacture porous structures, a positive raster fill gap is applied to create channels within the build layer. The channels are regularly arranged and interconnected in three dimensions. Layer-by-layer fabrication allows for the design of varying pore morphologies across the scaffold structure.

[0088] In some embodiments, the method includes selective laser sintering (SLS). Selective laser sintering (SLS) is a process in which a dispenser deposits layers of powdered material onto a target area. A laser control mechanism, typically comprising a computer having an article design stored thereon, is present. The laser control mechanism modulates and moves a laser beam to selectively irradiate powder layers within the defined boundaries of the design, causing the laser beam to melt such powder. This is done to selectively sinter successive powder layers. The method produces a finished product comprised of multiple layers sintered together.

[0089] In some embodiments, after 3D printing, the resulting object is immersed in water to dissolve certain components of the ink, such as PEG, particulates (e.g., pore-forming agents, sucrose), effervescent agents (e.g., sodium bicarbonate), or combinations thereof. The structure may then be dried, sterilized, treated with a therapeutic agent as described elsewhere herein, or a combination thereof.

[0090] Any of the 3D printed structures described herein can be coated with a tetherable protein (e.g., tBMP2). After completing the structure using any of the methods discussed herein, the structure can be washed in an acidic sodium acetate buffer. This can be one, two, or more washes. After washing, the structure can then be incubated for 2 hours in a sodium acetate buffer containing tBMP2 protein at a concentration of 1 mg / mL. The tetherable tBMP2 binds to the β-TCP surface of the single-layer implantable structure.

[0091] In further embodiments, the ink formulations discussed herein may include a photosensitive resin mixed with ceramic powder for digital light processing (DLP), an additive manufacturing technique faster than robocasting or melt extrusion. Components in photosensitive ceramic-filled resins for DLP 3D printing of bone implants typically include ceramic powder (e.g., β-TCP, hydroxyapatite, bioglass, typically with particle sizes of 10 μm or less), one or more crosslinked acrylates or methacrylates (e.g., polyethylene glycol diacrylate, polycaprolactone methacrylate), a plasticizer (e.g., water) to reduce resin viscosity, a dispersant (e.g., Darvan® 821-A) to promote powder agglomeration breakup, a photoinitiator (e.g., lithium phenyl-2,4,6-trimethylbenzoylphosphinate) to initiate the photocrosslinking reaction, and a photoabsorber (e.g., tartrazine) to maintain high xy resolution. Once the resin formulation is prepared by asymmetric centrifugal mixing of the components, the ink is exposed layer by layer to a DLP image, which selectively solidifies the illuminated pixels when the resin is exposed to light. Once the implantable structure is built layer by layer, it can be heat-treated to sinter the included polymer and densify the ceramic (e.g., polyethylene glycol diacrylate-containing resin), or it can remain intact, resulting in a flexible ceramic / polymer composite implant (e.g., polycaprolactone methacrylate-containing resin).

[0092] device In another aspect, devices and kits are provided that include the 3D printed structures described herein and therapeutic agents. In some embodiments, the devices include therapeutic agents that are bonded to, dispersed within, or otherwise combined with the 3D printed structures. As used herein, a therapeutic agent includes multiple therapeutic agents, such as two, three, four, or five therapeutic agents.

[0093] Treatment drugs In some embodiments, the therapeutic agent comprises a mammalian growth factor or a functional portion thereof. The mammalian growth factor can be an osteoinductive molecule capable of initiating or enhancing the bone repair process. The functional portion of the mammalian growth factor is a region having a therapeutic effect. For example, the functional portion of the mammalian growth factor is osteoinductive. As another example, the functional portion of the mammalian growth factor can initiate and / or enhance bone repair. The functional portion of the mammalian growth factor can have osteogenic activity.

[0094] Non-limiting examples of mammalian growth factors are described herein. Mammalian growth factors include epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-I), fibroblast growth factor (FGF), fibroblast growth factor 2 (FGF2), fibroblast growth factor 18 (FGF18), transforming growth factor alpha (TGF-α), transforming growth factor beta (TGF-β), transforming growth factor beta-1 (TGF-β1), transforming growth factor beta-3 (TGF-β3), osteogenic protein 1 (OP-I), osteogenic protein 2 (OP-2), osteogenic protein 3 (OP-3), bone morphogenetic protein 2 (BMP), and the like. -2), bone morphogenetic protein 3 (BMP-3), bone morphogenetic protein 4 (BMP-4), bone morphogenetic protein 5 (BMP-5), bone morphogenetic protein 6 (BMP-6), bone morphogenetic protein 7 (BMP-7), bone morphogenetic protein 9 (BMP-9), bone morphogenetic protein 10 (BMP-10), bone morphogenetic protein 11 (BMP-11), bone morphogenetic protein 12 (BMP-12), bone morphogenetic protein 13 (BMP-13), bone morphogenetic protein 15 (BMP-15), dentin phosphoprotein (DPP), plant-related growth factor (Vgr), growth differentiation factor 1 (GD) F-1), growth differentiation factor 3 (GDF-3), growth differentiation factor 5 (GDF-5), growth differentiation factor 6 (GDF-6), growth differentiation factor 7 (GDF-7), growth differentiation factor 8 (GDF8), growth differentiation factor 11 (GDF11), growth differentiation factor 15 (GDF15), vascular endothelial growth factor (VEGF), hyaluronic acid-binding protein (HABP), collagen-binding protein (CBP), fibroblast growth factor 18 (FGF-18), keratinocyte growth factor (KGF), tumor necrosis factor α (TNFα), tumor necrosis factor (TNF)-related apoptosis-inducing ligase (ATLI). and (TRAIL), wnt family member 1 (WNT1), wnt family member 2 (WNT2), wnt family member 2B (WNT2B), wnt family member 3 (WNT3), wnt family member 3A (WNT3A), wnt family member 4 (WNT4), wnt family member 5A (WNT5A), wnt family member 5B (WNT5B), wnt family member 6 (WNT6), wnt family member 7A (WNT7A), wnt family member 7B (WNT7B),wnt family member 8A (WNT8A), wnt family member 8B (WNT8B), wnt family member 9A (WNT9A), wnt family member 9B (WNT9B), wnt family member 10A (WNT10A), wnt family member 10B (WNT10B), wnt family member 11 (WNT11), or wnt family member 16 (WNT16), or a mature peptide or functional portion thereof.

[0095] In some embodiments, the mammalian growth factor is a human growth factor. Non-limiting examples of human growth factors and mature peptides and / or functional portions thereof are provided in Table 1. In some embodiments, the mammalian growth factor comprises a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical) to any of the sequences in Table 1 or to any secreted human growth factor and has osteogenic activity. In some embodiments, amino acids in mammalian growth factors that are conserved among various species may be important for osteogenic activity and may not be mutated, while amino acids in mammalian growth factors that are not conserved among various species may be important for osteogenic activity and may be mutated.

[0096] In some embodiments, the mammalian growth factor comprises BMP-2. In some embodiments, the mammalian growth factor is a mature peptide of BMP-2 (e.g., without the signal sequence). In some embodiments, the mammalian growth factor comprises a functional portion of BMP-2. In some embodiments, the functional portion of BMP-2 comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR (SEQ ID NO: 454). In some embodiments, the mammalian growth factor comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 454. In some embodiments, the mammalian growth factor comprises a sequence at least about 90% identical to SEQ ID NO: 454. In some embodiments, the mammalian growth factor comprises SEQ ID NO: 454.

[0097] In some embodiments, the mammalian growth factor is a non-human mammalian growth factor. The non-human mammalian growth factor can be homologous to a human growth factor, such as one or more of the human growth factors in Table 1. In some embodiments, a non-human mammalian growth factor is homologous to a human growth factor if the non-human mammalian growth factor is at least about 80% identical to the human mammalian growth factor as determined using the NCBI Blast alignment algorithm as of the date of this application. In some cases, the coverage is at least about 90%. In some embodiments, a non-human mammalian growth factor is homologous to a human growth factor if the non-human mammalian growth factor is at least about 80% positive to the human mammalian growth factor as determined using the NCBI Blast alignment algorithm as of the date of this application. In some cases, the coverage is at least about 90%. In some embodiments, a non-human mammalian growth factor is homologous to a human growth factor if, as of the filing date of this application, the non-human mammalian growth factor aligns to the human growth factor using NCBI Blast has an E-value of about 1E-40, at least about 1E-50, 1E-60, 1E-70, or less than 1E-10, with a query coverage of at least about 90%.

[0098] [Table 1-1]

[0099] [Table 1-2]

[0100] [Table 1-3]

[0101] [Table 1-4]

[0102] [Table 1-5]

[0103] [Table 1-6]

[0104] targeting part In some embodiments, the device or kit includes a targeting moiety that tethers the therapeutic agent to the structure. In some embodiments, the targeting moiety is attached to the therapeutic agent, and the targeting moiety is non-covalently attached to the structure. As a non-limiting example, the targeting moiety is covalently attached to the therapeutic agent via a peptide bond. For example, the targeting moiety includes a targeting peptide, and the targeting peptide is attached to the therapeutic agent via a peptide bond.

[0105] In some embodiments, the targeting moiety has an affinity for the structure, or a component of the structure, e.g., the ceramic material of the structure, such as calcium phosphate. In some embodiments, the dissociation constant (KD) for binding between the targeting moiety and the structure or a component thereof is (i) at least about 1 fM, at least about 10 fM, at least about 100 fM, or at least about 1 pM; and (ii) less than about 100 μM, less than about 90 μM, less than about 80 μM, less than about 70 μM, less than about 60 μM, less than about 50 μM, less than about 40 μM, less than about 30 μM, less than about 20 μM, less than about 10 μM, less than about 5 μM, less than about 1 μM, or less than about 100 pM. For example, the targeting moiety may bind to β-tricalcium phosphate with an affinity of about 100 fM to about 100 μM, about 1 pM to about 100 μM, about 10 pM to about 100 μM, about 100 pM to about 100 μM, or about 1 μM to about 100 μM.

[0106] In some embodiments, the targeting moiety comprises one or more targeting peptides, each of which binds to the structure. In some embodiments, the targeting peptide binds to the ceramic material of the structure. For example, the targeting peptide binds to calcium phosphate (e.g., tricalcium phosphate, beta tricalcium phosphate, alpha tricalcium phosphate), hydroxyapatite, fluorapatite, bone (e.g., demineralized bone), glass (bioglass), such as silicate, vanadate, and related ceramic minerals, or chelated divalent metal ions, or combinations thereof. In some embodiments, the targeting peptide comprises two or more targeting peptides. In some embodiments, the two or more targeting peptides are no more than about 50, 45, 40, 35, 30, 25, 20, 15, or 10 targeting peptides. In some embodiments, the two or more targeting peptides are no more than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 targeting peptides. In some embodiments, the two or more targeting peptides are about 2 to about 10 targeting peptides. In some embodiments, the two or more targeting peptides are about 5 targeting peptides.

[0107] In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 1. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 2. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 3. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 4. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 5. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 6. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 7. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 8. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 9. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 10. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 11. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 12. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 13. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 14.In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 15. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 16. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 17. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 18. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 19. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 20. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 21. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 22. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 23. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 24. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 25. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 26. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 27.In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 28. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 29. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 30. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 31. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 32. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 33. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 34. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 35. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 36. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 37. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 38. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 39. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 40.In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 41. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 42. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 43. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 44. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 45. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 46. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 47. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 48. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 49. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 50. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 51. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 52. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 53.In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 54. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 55. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 56. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 57. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 58. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 59. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 60. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 61.

[0108] [Table 2-1]

[0109] [Table 2-2]

[0110] [Table 2-3]

[0111] In some embodiments, the targeting peptide comprises one or more sequences in Table 2. In some embodiments, the targeting peptide comprises a sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to a sequence in Table 2.

[0112] [Table 3-1]

[0113] [Table 3-2]

[0114] [Table 3-3]

[0115] [Table 3-4]

[0116] [Table 3-5]

[0117] In some embodiments, the targeting peptide comprises one or more sequences in Table 3. In some embodiments, the targeting peptide comprises a sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to a sequence in Table 3.

[0118] Additional targeting peptides useful in the present disclosure include any one of SEQ ID NOs: 1 through 558 of U.S. Patent No. 7,572,766. In some embodiments, the targeting peptide comprises a sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to any one of SEQ ID NOs: 1 through 558 of U.S. Patent No. 7,572,766.

[0119] In some embodiments, the device or kit comprises a chimeric polypeptide comprising a targeting peptide and a targeting moiety. In some cases, the chimeric polypeptide comprises at least about 70%, 75%, 80%, 85%, 90%, 95% or 100% identical sequence to SEQ ID NO: 433 (ASGAGGSEGGGSEGGTSGATGAGTSTSGGGASTGGGTGQAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR). In some cases, the chimeric polypeptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 434 (MPIGSLLADTTHHRPWTVIGESTHHRPWSIIGESSHHKPFTGLGDTTHHRPWGILAESTHHKPWTASGAGGSEGGGSEGGTSGATGAGTSTSGGGASTGGGTGQAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR). In some cases, the chimeric polypeptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 435 (LLADTTHHRPWTVIGESTHHRPWSIIGESSHHKPFTGLGDTTHHRPWGILAESTHHKPWTASGAGGSEGGGSEGGTSGATGAGTSTSGGGASTGGGTGQAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR).In some cases, the chimeric polypeptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 436 (VIGESTHHRPWSIIGESSHHKPFTGLGDTTHHRPWGILAESTHHKPWTASGAGGSEGGGSEGGTSGATGAGTSTSGGGASTGGGTGQAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR). In some cases, the chimeric polypeptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 437 (IIGESSHHKPFTGLGDTTHHRPWGILAESTHHKPWTASGAGGSEGGGSEGGTSGATGAGTSTSGGGASTGGGTGQAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR). In some cases, the chimeric polypeptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 438 (GLGDTTHHRPWGILAESTHHKPWTASGAGGSEGGGSEGGTSGATGAGTSTSGGGASTGGGTGQAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR).In some cases, the chimeric polypeptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 439 (ILAESTHHKPWTASGAGGSEGGGSEGGTSGATGAGTSTSGGGASTGGGTGQAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR). In some cases, the chimeric polypeptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 440 ((X)QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR), where X comprises a targeting peptide and, optionally, a linker. For example, the targeting peptide comprises one or more of SEQ ID NOs: 1-41. In some cases, the chimeric polypeptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 441 ((X)ASGAGGSEGGGSEGGTSGATGAGTSTSGGGASTGGGTGQAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR), where X comprises a targeting peptide and, optionally, a linker. For example, the targeting peptide comprises one or more of SEQ ID NOs: 1-41.

[0120] In some embodiments, the therapeutic agent is not bound to the structure using a targeting moiety. For example, the therapeutic agent may interact with the structure through non-covalent bonding. The therapeutic agent may be bound to the structure by hydrogen bonding, ionic bonding, hydrophobic interaction, or van der Waals force. The therapeutic agent may also be bound to the structure using covalent bonding. Examples of methods for binding using covalent bonding include chemical linkers and spacers used to modify active groups in proteins, such as amines, thiols, and carbohydrates.

[0121] In some embodiments, a device is provided that includes a cell-seeded structure. Non-limiting examples of cells include osteocytes and other bone cells, chondrocytes, and meniscus cells. In some instances, the cells may be added to a completed implantable structure.

[0122] Device fabrication Further provided herein are methods for manufacturing a device comprising a structure (e.g., a scaffold) and a therapeutic agent. Some methods include (a) providing a first solution of a therapeutic agent (e.g., a chimeric polypeptide comprising a therapeutic agent and a targeting moiety), (b) providing a 3D structure, and (c) combining (a) and (b). In some embodiments, the method further includes (d) washing the 3D structure of step (c) with a second solution, such as phosphate-buffered saline (PBS). In some embodiments, the method further includes drying the 3D structure of step (c) or step (d).

[0123] In some embodiments, the mass of therapeutic agent (e.g., therapeutic agent alone or therapeutic agent conjugated to a targeting moiety) per cubic centimeter of structure within the device is between about 0.05 and 50 mg / cc, e.g., about 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mg / cc, or any value therebetween. For example, the therapeutic agent is about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 mg per cubic centimeter of device. One method for determining the amount of therapeutic peptide bound to the structure includes (1) measuring the mass of the therapeutic peptide input in a first solution, (2) measuring the mass of the therapeutic agent remaining in the first solution after combination with and removal from the structure, (3) if a washing step is included, measuring the mass of the therapeutic agent in a second solution, and (4) summing (2) and (3) and subtracting the sum of (4) from (1).

[0124] Treatment method In another aspect, methods of treating a subject with the structure herein are provided. In some methods, the subject is treated with a device comprising a therapeutic peptide and the structure. In some examples, the subject has a fracture or bone defect. In some examples, the subject is in need of vertebral fusion of the spine. In some examples, the subject has a cartilage tear or cartilage defect. In some examples, the subject has chondropenia.

[0125] In some embodiments, the subject suffers from a defect of bone, cartilage, soft tissue, tendon, fascia, ligament, organ, bone-tendon tissue, skin, or osteochondral, or a combination of one or more of the foregoing defects. In some embodiments, the defect is a lack of bone, cartilage, soft tissue, tendon, fascia, ligament, organ, bone-tendon tissue, skin, or osteochondral, or a combination of one or more of the foregoing defects. In some embodiments, the subject's defect results from trauma. In some embodiments, the subject's defect results from a congenital disease. In some embodiments, the subject's defect results from an acquired disease. In some embodiments, the defect refers to the absence, loss, and / or damage of bodily tissue and / or organs. In some embodiments, a "bone defect" refers to the absence or loss (e.g., partial loss) of bone at a subject's anatomical location that would otherwise be present in a healthy subject. Bone defects may be the result of infection (e.g., osteomyelitis), tumor, trauma, or adverse events from a treatment. Bone defects may also affect and cause damage to the muscles, soft tissues, tendons, or joints surrounding the bone defect. In some embodiments, bone defect includes damage to soft tissue. In some embodiments, "cartilage defect" refers to the absence or reduction (e.g., partial reduction) of cartilage at an anatomical location of a subject that would otherwise be present in a healthy subject. Cartilage defect may be the result of disease, osteochondritis, osteonecrosis, or trauma. For example, cartilage defect may affect the knee joint.

[0126] Non-limiting examples of diseases suitable for treatment with the structures or devices described herein include osteoarthritis, disc degeneration, congenital defects, spinal stenosis, spondylolisthesis, spondylosis, fractures, scoliosis, kyphosis, spinal fusion (PLF and interbody fusion), bone trauma repair, dental repair, craniomaxillofacial repair, ankle fusion, vertebroplasty, balloon osteoplasty, navicular fracture repair, tendon-bone repair, osteoporosis, avascular necrosis, congenital skeletal deformities, rib reconstruction, subchondral bone repair, cartilage repair (e.g., at low doses), or trauma, or a combination thereof. BMP2 is also involved in the development of hair follicles, and therefore, methods may include treatment of hair follicles. Trauma may be to bone, cartilage, soft tissue, tendon, fascia, ligament, organ, bone-tendon tissue, or skin tissue, or osteochondral tissue. In some embodiments, the method is for treating osteochondral injuries.

[0127] The treatment method may include spinal fusion. In some embodiments, spinal fusion is a surgical procedure to join two or more vertebrae. In some embodiments, the spinal fusion includes PLF. In some embodiments, the spinal fusion includes interbody fusion.

[0128] Provided herein are methods for promoting bone or cartilage formation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any of the structures or devices described herein. Some embodiments of these methods may further comprise first selecting a subject in need of bone or cartilage formation. In some embodiments, the structure or device is administered to the subject proximal to a desired site of bone or cartilage formation in the subject.

[0129] Also provided herein are methods of replacing and / or repairing bone or cartilage in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any of the structures or devices described herein. Some embodiments of these methods may further comprise first selecting a subject in need of bone replacement, bone repair, cartilage replacement, or cartilage repair. In some embodiments, the structure or device is administered to the subject proximal to the desired site of bone or cartilage replacement or repair in the subject.

[0130] Also provided herein are methods for treating a fracture or osteopenia in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any of the structures or devices described herein. Some embodiments of these methods may further comprise first selecting a subject with a fracture or osteopenia. In some embodiments, the structure or device is administered to the subject proximal to the fracture or osteopenia site in the subject.

[0131] Also provided herein are methods of repairing soft tissue in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any of the structures or devices described herein. Some embodiments of these methods may further comprise first selecting a subject with a fracture or osteopenia. In some embodiments, the composition is administered to the subject proximal to the fracture or site of osteopenia in the subject.

[0132] Also provided herein are methods for locally delivering a therapeutic agent to a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any of the structures or devices described herein. Some embodiments of these methods may further comprise first selecting a subject with a fracture or osteopenia. In some embodiments, the structure or device is administered to the subject proximal to a fracture or site of osteopenia in the subject.

[0133] Methods for determining the effectiveness of treating a fracture or osteopenia in a subject are known in the art and include, for example, imaging techniques (eg, magnetic resonance imaging, X-ray, or computed tomography).

[0134] Methods for detecting bone or cartilage formation, or bone or cartilage replacement or repair in a subject are also known in the art and include, for example, imaging techniques (e.g., magnetic resonance imaging, X-ray, or computed tomography).

[0135] The animal model suitable for treating fracture or osteopenia, forming bone or cartilage, or replacing or repairing bone or cartilage is known in the art.Non-limiting examples of such animal model are described in Examples and, for example, Drosse et al., Tissue Engineering Part C 14(1):79-88,2008; Histing et al., Bone 49:591-599,2011; and Poser et al., Hindawi Publishing Corporation, BioMed Research International; Article ID 348635,2014.

[0136] As used herein, a method of treatment includes administering a structure or device herein to a subject. In some embodiments, the administering includes implanting a polypeptide or composition herein.

[0137] In some embodiments, a polypeptide and / or composition herein comprising BMP-2 is administered to a subject. In some embodiments, the BMP2 comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 454. In some embodiments, the BMP-2 is administered to induce bone formation in a subject. In some embodiments, the BMP-2 is administered to induce cartilage formation. In some embodiments, the BMP-2 is administered in spinal fusion surgery.

[0138] The terms used herein are for descriptive purposes only and are not intended to be limiting. The singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. To the extent that the terms "including," "includes," "having," "has," "with," or variations thereof are used in either the detailed description and / or claims, such terms are intended to be inclusive in the same manner as the term "comprising."

[0139] In some embodiments, the term "about" or "approximately" means within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, as is customary with any value.

[0140] As used herein, the term "subject" refers to any mammal. Thus, a subject refers to, for example, a mouse, a rat, a dog, a cat, a horse, a cow, a pig, a guinea pig, a rat, a human, a monkey, etc. When the subject is a human, the subject may be referred to herein as a patient. In some embodiments, the subject or "subject in need of treatment" may be a canine (e.g., dog), feline (e.g., cat), equine (e.g., horse), ovine, bovine, porcine, caprine, primate, such as a simian (e.g., a monkey (e.g., marmoset, baboon), or ape (e.g., gorilla, chimpanzee, orangutan, or gibbon), human, or rodent (e.g., mouse, guinea pig, hamster, or rat). In some embodiments, the subject or "subject in need of treatment" may be a non-human mammal, particularly a mammal traditionally used as a model for demonstrating therapeutic efficacy in humans (e.g., a rat, rabbit, pig, canine, or primate).

[0141] In some embodiments, the term "therapeutically effective amount" refers to an amount of a polypeptide or composition effective to "treat" a disease, condition, or disorder in a subject. In some cases, a therapeutically effective amount of a polypeptide or composition reduces the severity of symptoms of the disease, condition, or disorder. In some instances, the disease, condition, or disorder involves organ or tissue deficiency.

[0142] In some embodiments, "affinity" refers to the strength of the sum of non-covalent interactions between a β-TCP binding sequence (or a chimeric polypeptide or polypeptide containing a β-TCP binding sequence) and its binding partner (e.g., β-TCP). Affinity can be measured by common methods known in the art, including those described herein. Affinity can be determined, for example, using surface plasmon resonance (SPR) technology (e.g., BIACORE®) or biolayer interferometry (e.g., FORTEBIO®).

[0143] The percent (%) sequence identity to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and any conservative substitutions are not considered as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in a variety of known ways, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences, including the algorithm required to achieve maximum alignment over the entire length of the sequences being compared, can be determined. However, for the purposes herein, the percent amino acid sequence identity value is generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and its source code, together with user documentation, has been submitted to the U.S. Copyright Office (Washington, DC, 20559) and is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, California) or can be compiled from source code. The ALIGN-2 program must be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0144] In situations where ALIGN-2 is utilized for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A with, to, or against a given amino acid sequence B (which may alternatively be expressed as a given amino acid sequence A having or containing a certain % amino acid sequence identity with, to, or against a given amino acid sequence B) is calculated as follows: multiply the fraction X / Y by 100, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in its alignment of A and B, and where Y is the total number of amino acid residues in B. It should be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A. Unless specifically specified otherwise, all % amino acid sequence identity values ​​used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0145] Further embodiments Embodiment 1. A three-dimensional structure comprising about 70% to about 80% by weight of β-TCP and about 20% to about 30% by weight of polycaprolactone (PCL). Embodiment 2. The three-dimensional structure of embodiment 1, comprising about 75% by weight of βTCP and about 25% by weight of PCL. Embodiment 3. A three-dimensional structure comprising about 70% to about 80% by weight of β-TCP and about 20% to about 30% by weight of a caprolactone / glycolide copolymer. Embodiment 4. The three-dimensional structure of embodiment 3, comprising about 75% by weight of β-TCP and about 25% by weight of caprolactone / glycolide copolymer. Embodiment 5. The three-dimensional structure of embodiment 3 or embodiment 4, wherein the caprolactone / glycolide copolymer is caprolactone / glycolide copolymer (95:5). Embodiment 6. The three-dimensional structure of embodiment 3 or embodiment 4, wherein the caprolactone / glycolide copolymer is a caprolactone / glycolide copolymer (90:10). Embodiment 7. A three-dimensional structure comprising about 70% to about 80% by weight of β-TCP and about 20% to about 30% by weight of poly(D,L-lactide-co-glycolide) copolymer. Embodiment 8. The three-dimensional structure of embodiment 7, comprising about 75% by weight of βTCP and about 25% by weight of poly(D,L-lactide-co-glycolide) copolymer. Embodiment 9. The three-dimensional structure of embodiment 7 or embodiment 8, wherein the caprolactone / glycolide copolymer is poly(D,L-lactide-co-glycolide) copolymer (50:50). Embodiment 10. A three-dimensional structure comprising about 70% to about 80% by weight of β-TCP and about 20% to about 30% by weight of polydioxanone (PDS). Embodiment 11. The three-dimensional structure of embodiment 10, comprising about 75% by weight of βTCP and about 25% by weight of PDS. Embodiment 12. A three-dimensional structure comprising about 70% to about 80% by weight of β-TCP and about 20% to about 30% by weight of dioxanone / L-lactide copolymer. Embodiment 13. The three-dimensional structure of embodiment 12, comprising about 75% by weight of β-TCP and about 25% by weight of dioxanone / L-lactide copolymer. Embodiment 14. The three-dimensional structure of embodiment 12 or embodiment 13, wherein the caprolactone / glycolide copolymer is a dioxanone / L-lactide copolymer (90:10). Embodiment 15. A three-dimensional structure comprising about 70% to about 80% by weight of β-TCP and about 20% to about 30% by weight of glycolide / L-lactide copolymer. Embodiment 16. The three-dimensional structure of embodiment 15, comprising about 75% by weight of β-TCP and about 25% by weight of glycolide / L-lactide copolymer. Embodiment 17. The three-dimensional structure of embodiment 15 or embodiment 16, wherein the caprolactone / glycolide copolymer is a glycolide / L-lactide copolymer (95:5). Embodiment 18. A three-dimensional structure comprising about 70% to about 80% by weight of β-TCP and about 20% to about 30% by weight of poly-l-lactide. Embodiment 19. The three-dimensional structure of embodiment 18, comprising about 75% by weight of βTCP and about 25% by weight of poly-l-lactide. Embodiment 20. The three-dimensional structure according to any one of embodiments 1 to 19, having a density of about 1 g / cm 3 to about 1.5 g / cm 3 . Embodiment 21. The three-dimensional structure according to any one of embodiments 1 to 20, having an open porosity of about 25% to about 40%. Embodiment 22. A three-dimensional structure described in any one of embodiments 1 to 21, wherein the strut diameter is about 300 μm to 800 μm, or about 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, or 800 μm. Embodiment 23. A three-dimensional structure according to any one of embodiments 1 to 20, comprising a plurality of micropores, the average pore size of the micropores being from about 1 micron to about 500 microns, or from about 1 micron to about 50 microns. Embodiment 24. A method for preparing a three-dimensional structure according to any one of embodiments 1 to 22, the method comprising additive manufacturing. Embodiment 25. The method of embodiment 24, wherein the additive manufacturing comprises fused granule manufacturing (FGF) or fused filament manufacturing (FFF). Embodiment 26. An ink formulation comprising about 55% to about 65% by weight β-TCP, about 15% to about 25% by weight PCL, about 5% to about 15% PEG having a molecular weight of about 500 g / mol to about 15,000 g / mol, and about 5% to about 15% PEG having a molecular weight of about 25,000 g / mol to about 50,000 g / mol. Embodiment 27. The ink formulation of embodiment 26, comprising about 60% by weight β-TCP, about 20% by weight PCL, about 10% by weight PEG having a molecular weight of about 500 g / mol to about 15,000 g / mol, and about 10% PEG having a molecular weight of about 25,000 g / mol to about 50,000 g / mol. Embodiment 28. The ink formulation of embodiment 27, comprising about 60% by weight of βTCP, about 20% by weight of PCL, about 10% by weight of PEG having a molecular weight of about 8,000 g / mol, and about 10% by weight of PEG having a molecular weight of about 35,000 g / mol. Embodiment 29. The ink formulation of any one of embodiments 26 to 28, comprising about 1% to about 10% of a sacrificial pore-forming agent. Embodiment 30. The ink formulation of embodiment 29, wherein the sacrificial pore-forming agent comprises sucrose. Embodiment 31. An ink formulation comprising about 55% to about 65% by weight β-TCP, about 15% to about 25% by weight caprolactone / glycolide copolymer, about 5% to about 15% PEG having a molecular weight of about 500 g / mol to about 15,000 g / mol, and about 5% to about 15% PEG having a molecular weight of about 25,000 g / mol to about 50,000 g / mol. Embodiment 32. The ink formulation of embodiment 31, comprising about 60% by weight of β-TCP, about 20% by weight of caprolactone / glycolide copolymer, about 10% by weight of PEG having a molecular weight of about 500 g / mol to about 15,000 g / mol, and about 10% by weight of PEG having a molecular weight of about 25,000 g / mol to about 50,000 g / mol. Embodiment 33. The ink formulation of embodiment 32, comprising about 60% by weight of β-TCP, about 20% by weight of caprolactone / glycolide copolymer, about 10% by weight of PEG having a molecular weight of about 8,000 g / mol, and about 10% by weight of PEG having a molecular weight of about 35,000 g / mol. Embodiment 34. The ink formulation of any one of embodiments 31 to 33, wherein the caprolactone / glycolide copolymer is caprolactone / glycolide copolymer (95:5). Embodiment 35. The ink formulation of any one of embodiments 31 to 33, wherein the caprolactone / glycolide copolymer is a 90:10 caprolactone / glycolide copolymer. Embodiment 36. An ink formulation comprising about 55% to about 65% by weight β-TCP, about 15% to about 25% by weight poly(D,L-lactide-co-glycolide) copolymer, about 5% to about 15% PEG having a molecular weight of about 500 g / mol to about 15,000 g / mol, and about 5% to about 15% PEG having a molecular weight of about 25,000 g / mol to about 50,000 g / mol. Embodiment 37. The ink formulation of embodiment 36, comprising about 60% by weight of β-TCP, about 20% by weight of poly(D,L-lactide-co-glycolide) copolymer, about 10% by weight of PEG having a molecular weight of about 500 g / mol to about 15,000 g / mol, and about 10% by weight of PEG having a molecular weight of about 25,000 g / mol to about 50,000 g / mol. Embodiment 38. The ink formulation of embodiment 37, comprising about 60% by weight of βTCP, about 20% by weight of poly(D,L-lactide-co-glycolide) copolymer, about 10% by weight of PEG having a molecular weight of about 8,000 g / mol, and about 10% by weight of PEG having a molecular weight of about 35,000 g / mol. Embodiment 39. The ink formulation of any one of embodiments 36 to 38, wherein the caprolactone / glycolide copolymer is a 50:50 caprolactone / glycolide copolymer. Embodiment 40. An ink formulation comprising about 55% to about 65% by weight β-TCP, about 15% to about 25% by weight PDS, about 5% to about 15% PEG having a molecular weight of about 500 g / mol to about 15,000 g / mol, and about 5% to about 15% PEG having a molecular weight of about 25,000 g / mol to about 50,000 g / mol. Embodiment 41. The ink formulation of embodiment 40, comprising about 60% by weight of β-TCP, about 20% by weight of PDS, about 10% by weight of PEG having a molecular weight of about 500 g / mol to about 15,000 g / mol, and about 10% by weight of PEG having a molecular weight of about 25,000 g / mol to about 50,000 g / mol. Embodiment 42. The ink formulation of embodiment 41, comprising about 60% by weight of βTCP, about 20% by weight of PDS, about 10% by weight of PEG having a molecular weight of about 8,000 g / mol, and about 10% by weight of PEG having a molecular weight of about 35,000 g / mol. Embodiment 43. An ink formulation comprising about 55% to about 65% by weight β-TCP, about 15% to about 25% by weight dioxanone / L-lactide copolymer, about 5% to about 15% PEG having a molecular weight of about 500 g / mol to about 15,000 g / mol, and about 5% to about 15% PEG having a molecular weight of about 25,000 g / mol to about 50,000 g / mol. Embodiment 44. The ink formulation of embodiment 43, comprising about 60% by weight of β-TCP, about 20% by weight of dioxanone / L-lactide copolymer, about 10% by weight of PEG having a molecular weight of about 500 g / mol to about 15,000 g / mol, and about 10% by weight of PEG having a molecular weight of about 25,000 g / mol to about 50,000 g / mol. Embodiment 45. The ink formulation of embodiment 44, comprising about 60% by weight of βTCP, about 20% by weight of dioxanone / L-lactide copolymer, about 10% by weight of PEG having a molecular weight of about 8,000 g / mol, and about 10% by weight of PEG having a molecular weight of about 35,000 g / mol. Embodiment 46. The ink formulation of any one of embodiments 43 to 45, wherein the caprolactone / glycolide copolymer is a 90:10 caprolactone / glycolide copolymer. Embodiment 47. An ink formulation comprising about 55% to about 65% by weight β-TCP, about 15% to about 25% by weight glycolide / L-lactide copolymer, about 5% to about 15% PEG having a molecular weight of about 500 g / mol to about 15,000 g / mol, and about 5% to about 15% PEG having a molecular weight of about 25,000 g / mol to about 50,000 g / mol. Embodiment 48. The ink formulation of embodiment 47, comprising about 60% by weight of β-TCP, about 20% by weight of glycolide / L-lactide copolymer, about 10% by weight of PEG having a molecular weight of about 500 g / mol to about 15,000 g / mol, and about 10% by weight of PEG having a molecular weight of about 25,000 g / mol to about 50,000 g / mol. Embodiment 49. The ink formulation of embodiment 48, comprising about 60% by weight of β-TCP, about 20% by weight of glycolide / L-lactide copolymer, about 10% by weight of PEG having a molecular weight of about 8,000 g / mol, and about 10% by weight of PEG having a molecular weight of about 35,000 g / mol. Embodiment 50. The ink formulation of any one of embodiments 47 to 49, wherein the caprolactone / glycolide copolymer is glycolide / L-lactide copolymer (95:5). Embodiment 51. An ink formulation comprising about 55% to about 65% by weight β-TCP, about 15% to about 25% by weight poly-l-lactide, about 5% to about 15% PEG having a molecular weight of about 500 g / mol to about 15,000 g / mol, and about 5% to about 15% PEG having a molecular weight of about 25,000 g / mol to about 50,000 g / mol. Embodiment 52. The ink formulation of embodiment 51, comprising about 60% by weight of β-TCP, about 20% by weight of poly-l-lactide, about 10% by weight of PEG having a molecular weight of about 500 g / mol to about 15,000 g / mol, and about 10% by weight of PEG having a molecular weight of about 25,000 g / mol to about 50,000 g / mol. Embodiment 53. The ink formulation of embodiment 52, comprising about 60% by weight of βTCP, about 20% by weight of poly-l-lactide, about 10% by weight of PEG having a molecular weight of about 8,000 g / mol, and about 10% by weight of PEG having a molecular weight of about 35,000 g / mol. Embodiment 54. A method for preparing a three-dimensional structure, comprising additive manufacturing using an ink formulation according to any one of embodiments 26 to 53. Embodiment 55. The method of embodiment 54, wherein the ink formulation is in the form of pellets. Embodiment 56. The method of embodiment 55, wherein the additive manufacturing comprises fused granulation (FGF). Embodiment 57. The method of embodiment 54, wherein the ink formulation is in the form of a filament. Embodiment 58. The method of embodiment 57, wherein the additive manufacturing comprises fused filament fabrication (FFF). Embodiment 59. A device comprising a therapeutic agent and a structure described in any one of embodiments 1 to 25 or embodiments 54 to 58. Embodiment 60. The device of embodiment 59, wherein the therapeutic agent is non-covalently bound to the structure. Embodiment 61. A device described in embodiment 59 or embodiment 60, wherein the therapeutic agent comprises a growth factor. Embodiment 62. The device of embodiment 61, wherein the growth factor is selected from Table 1. Embodiment 63. A device described in any one of embodiments 59 to 62, wherein the therapeutic agent comprises a bone morphogenetic protein (BMP). Embodiment 64. A device described in any one of embodiments 59 to 63, wherein the therapeutic agent comprises a targeting moiety, and the targeting moiety is non-covalently bound to the structure. Embodiment 65. The device of embodiment 64, wherein the targeting moiety comprises a polypeptide at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to any one of the sequences in Tables 2-3. Embodiment 66. A device described in any one of embodiments 59 to 65, wherein the therapeutic agent comprises a chimeric polypeptide comprising a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to any one of SEQ ID NOs: 433 to 441. Embodiment 67. A method for treating a bone defect in a subject in need thereof, comprising applying a structure described in any one of embodiments 1 to 25 or embodiments 54 to 58 to the bone defect of the subject. Embodiment 68. A method for treating a bone defect in a subject in need thereof, comprising applying a device described in any one of embodiments 59 to 66 to the bone defect of the subject. Embodiment 69 The method of embodiment 67 or embodiment 68, wherein the bone defect is in the spine.

[0146] Each of the embodiments described and illustrated herein has individual components and features which may be readily separated from or combined with the features of any of the other embodiments without departing from the scope or spirit of the invention. Methods described can be carried out in the order of events described or in any other order which is logically possible.

[0147] Many embodiments of the present disclosure have been described. Nevertheless, it will be understood that various modifications can be made without departing from the spirit and scope of the present disclosure. Moreover, while specific ink formulations are described, the specific amounts of each ink component can vary. Accordingly, other embodiments are within the scope of the following claims. [Example]

[0148] Example 1: Ink formulation and 3D printed scaffolds Ink formulation and 3D printed scaffold #1 This 3D printing ink material is a flexible polymer-ceramic composite containing β-TCP, which can be tethered to a therapeutic agent such as tBMP2. The formulation contains two sacrificial pore-forming agents (water-soluble polyethylene glycol and water-soluble sucrose) to expose a larger β-TCP surface area for tBMP2 binding. The ink is a low-viscosity formulation extruded through a 400 μm diameter nozzle of an Allevi 3 pneumatic bioprinter. After 3D printing, the resulting scaffold was immersed in water to dissolve the sacrificial polyethylene glycol and sucrose pore-forming agents. The resulting scaffold #1 contains 75 wt% β-TCP powder and 25 wt% polycaprolactone.

[0149] This ink can also be converted into a filament form and used to prepare 3D-printed scaffolds using a fused filament fabrication (FFF or FDM) 3D printer. After 3D printing, the resulting scaffold is immersed in water to dissolve the sacrificial polyethylene glycol and sucrose pore-forming agent. The resulting scaffold contains 75% by weight of β-TCP powder and 25% by weight of polycaprolactone.

[0150] The ink is also converted into pellets (eg, the filaments are formed into small pellets) and fed from a hopper to a mini-screw extrusion head, which melts the material and extrudes it through fine nozzles.

[0151] The ink is then freeze-milled into a fine powder, which is then heated with a laser using the SLS process to form the desired configuration.

[0152] [Table 4]

[0153] Ink Formulation #2 This 3D printing ink material is a flexible polymer-ceramic composite containing β-TCP, which can be tethered to therapeutic agents such as tBMP2. The formulation contains a sacrificial pore-forming agent (water-soluble polyethylene glycol) to expose a larger β-TCP surface area for drug binding. This formulation utilizes a copolymer of 95 mol% caprolactone and 5 mol% glycolide for faster bioresorption compared to polycaprolactone. This ink is a low-viscosity formulation extruded through a 320 μm diameter nozzle on an Allevi 3 pneumatic bioprinter. After 3D printing, the resulting scaffold was immersed in water to dissolve the sacrificial polyethylene glycol pore-forming agent. The resulting scaffold #2 contains 75 wt% β-TCP powder and 25 wt% caprolactone / glycolide copolymer (95:5).

[0154] This ink can also be converted into a filament form and utilized to prepare 3D-printed scaffolds using a fused filament fabrication (FFF or FDM) 3D printer. After 3D printing, the resulting scaffold is immersed in water to dissolve the sacrificial polyethylene glycol pore-forming agent. The resulting scaffold contains 75 wt% β-TCP powder and 25 wt% caprolactone / glycolide copolymer.

[0155] The ink is also converted into pellets (eg, the filaments are formed into small pellets) and fed from a hopper to a mini-screw extrusion head, which melts the material and extrudes it through fine nozzles.

[0156] The ink is then freeze-milled into a fine powder, which is then heated with a laser using the SLS process to form the desired configuration.

[0157] [Table 5]

[0158] Ink Formulation #3 This 3D printing ink material is a flexible polymer-ceramic composite containing β-TCP, which can be tethered to therapeutic agents such as tBMP2. The formulation contains a sacrificial pore-forming agent (water-soluble polyethylene glycol) to expose a larger β-TCP surface area for drug binding. The formulation utilizes 90 mol% caprolactone, 10 mol% glycolide copolymer for faster bioresorption compared to polycaprolactone. This ink is a low-viscosity formulation extruded through a 320 μm diameter nozzle on an Allevi 3 pneumatic bioprinter. After 3D printing, the resulting scaffold was immersed in water to dissolve the sacrificial polyethylene glycol pore-forming agent. The resulting scaffold #3 contains 75 wt% β-TCP powder and 25 wt% caprolactone / glycolide copolymer (90:10).

[0159] This ink can also be converted into a filament form and utilized to prepare 3D-printed scaffolds using a fused filament fabrication (FFF or FDM) 3D printer. After 3D printing, the resulting scaffold is immersed in water to dissolve the sacrificial polyethylene glycol pore-forming agent. The resulting scaffold contains 75 wt% β-TCP powder and 25 wt% caprolactone / glycolide copolymer (90:10).

[0160] The ink is also converted into pellets (eg, the filaments are formed into small pellets) and fed from a hopper to a mini-screw extrusion head, which melts the material and extrudes it through fine nozzles.

[0161] The ink is then freeze-milled into a fine powder, which is then heated with a laser using the SLS process to form the desired configuration.

[0162] [Table 6]

[0163] Ink Formulation #4 This 3D printing ink material is a flexible polymer-ceramic composite containing β-TCP, which can be tethered to therapeutic agents such as tBMP2. The formulation contains a sacrificial pore-forming agent (water-soluble polyethylene glycol) to expose a larger β-TCP surface area for drug binding. The formulation utilizes 50 mol%:50 mol% poly(D,L-lactide-co-glycolide) copolymer for its fast bioresorption properties compared to polycaprolactone. This ink is a low-viscosity formulation extruded through a 400 μm diameter nozzle on an Allevi 3 pneumatic bioprinter. After 3D printing, the resulting scaffold was immersed in water to dissolve the sacrificial polyethylene glycol pore-forming agent. The resulting scaffold #4 contains 75 wt% β-TCP powder and 25 wt% poly(D,L-lactide-co-glycolide) copolymer (50:50).

[0164] This ink can also be converted into a filament form and utilized to prepare 3D-printed scaffolds using a fused filament fabrication (FFF or FDM) 3D printer. After 3D printing, the resulting scaffold is immersed in water to dissolve the sacrificial polyethylene glycol pore-forming agent. The resulting scaffold contains 75 wt% β-TCP powder and 25 wt% poly(D,L-lactide-co-glycolide) copolymer (50:50).

[0165] The ink is also converted into pellets (eg, the filaments are formed into small pellets) and fed from a hopper to a mini-screw extrusion head, which melts the material and extrudes it through fine nozzles.

[0166] The ink is then freeze-milled into a fine powder, which is then heated with a laser using the SLS process to form the desired configuration.

[0167] [Table 7]

[0168] Ink Formulation #5 This 3D printing ink material is a flexible polymer-ceramic composite containing β-TCP, which can be tethered to therapeutic agents such as tBMP2. The formulation contains a sacrificial pore-forming agent (water-soluble polyethylene glycol) to expose a larger β-TCP surface area for drug binding. This ink is a medium-viscosity formulation molded into 1.75 mm filaments for 3D printing with a RepRap-style FFF 3D printer (e.g., Prusa i3 MK3S 3D printer) equipped with a 400 μm diameter nozzle. Higher molecular weight polyethylene glycol (8000 MW for FFF 3D printing and 1500 MW for syringe-based bioprinting) results in a more viscous material, which aids in the extrusion of 1.75 mm diameter filaments. After 3D printing, the resulting scaffold was immersed in water to dissolve the sacrificial polyethylene glycol pore-forming agent. The resulting scaffold #5 contains 75 wt% β-TCP powder and 25 wt% polycaprolactone.

[0169] The ink is also converted into pellets (eg, the filaments are formed into small pellets) and fed from a hopper to a mini-screw extrusion head, which melts the material and extrudes it through fine nozzles.

[0170] The ink is then freeze-milled into a fine powder, which is then heated with a laser using the SLS process to form the desired configuration.

[0171] [Table 8]

[0172] Ink Formulation #6 This 3D printing ink material is a flexible polymer-ceramic composite containing β-TCP, which can be tethered to therapeutic agents such as tBMP2. The formulation contains a sacrificial pore-forming agent (water-soluble polyethylene glycol) to expose a larger β-TCP surface area for drug binding. The ink also contains a foaming agent (sodium bicarbonate), which thermally decomposes during 3D printing to release CO2 gas and generate a foam structure, thereby increasing the porosity of the 3D-printed scaffold. This ink is a medium-viscosity formulation molded into 1.75 mm filaments for 3D printing with a RepRap-style FFF 3D printer (e.g., Prusa i3 MK3S 3D printer) equipped with a 400 μm diameter nozzle. Higher molecular weight polyethylene glycol (8000 MW for FFF 3D printing and 1500 MW for syringe-based bioprinting) results in a more viscous material that aids in the extrusion of 1.75 mm diameter filaments. After 3D printing, the resulting scaffold was immersed in water to dissolve the sacrificial polyethylene glycol pore former and sodium carbonate, a by-product of the thermal decomposition of sodium bicarbonate. The resulting scaffold #6 contains 75 wt% β-TCP powder and 25 wt% polycaprolactone.

[0173] The ink is also converted into pellets (eg, the filaments are formed into small pellets) and fed from a hopper to a mini-screw extrusion head, which melts the material and extrudes it through fine nozzles.

[0174] The ink is then freeze-milled into a fine powder, which is then heated with a laser using the SLS process to form the desired configuration.

[0175] [Table 9]

[0176] Ink Formulation #7 This 3D printing ink material is a flexible polymer-ceramic composite containing β-TCP, which can be tethered to therapeutic agents such as tBMP2. The formulation contains a sacrificial pore-forming agent (water-soluble polyethylene glycol) to expose a larger β-TCP surface area for drug binding. The formulation utilizes a 90 mol%:10 mol% poly(dioxanone-co-lactide) copolymer for faster bioresorption compared to polycaprolactone. This ink is a low-viscosity formulation that can be extruded through a 400 μm diameter nozzle on an Allevi 3 pneumatic bioprinter. After 3D printing, the resulting scaffold is immersed in water to dissolve the sacrificial polyethylene glycol pore-forming agent. The resulting scaffold contains 75 wt% β-TCP powder and 25 wt% dioxanone / L-lactide copolymer (90:10).

[0177] This ink can also be converted into a filament form and utilized to prepare 3D-printed scaffolds using a fused filament fabrication (FFF or FDM) 3D printer. After 3D printing, the resulting scaffold is immersed in water to dissolve the sacrificial polyethylene glycol pore-forming agent. The resulting scaffold contains 75 wt% β-TCP powder and 25 wt% dioxanone / L-lactide copolymer (90:10).

[0178] The ink is also converted into pellets (eg, the filaments are formed into small pellets) and fed from a hopper to a mini-screw extrusion head, which melts the material and extrudes it through fine nozzles.

[0179] The ink is then freeze-milled into a fine powder, which is then heated with a laser using the SLS process to form the desired configuration.

[0180] [Table 10]

[0181] Ink Formulation #8 This 3D printing ink material is a flexible polymer-ceramic composite containing β-TCP, which can be tethered to therapeutic agents such as tBMP2. The formulation contains a sacrificial pore-forming agent (water-soluble polyethylene glycol) to expose a larger β-TCP surface area for drug binding. This ink is a medium-viscosity formulation that can be molded into 1.75 mm filaments for 3D printing with a RepRap-style FFF 3D printer (e.g., the Prusa i3 MK3S 3D printer) equipped with a 400 μm diameter nozzle. Blends of higher molecular weight polyethylene glycol (8000 MW and 20,000 MW for FFF 3D printing, 1500 MW for syringe-based bioprinting) result in a more viscous material that aids in the extrusion of 1.75 mm diameter filaments. After 3D printing, the resulting scaffold is immersed in water to dissolve the sacrificial polyethylene glycol pore-forming agent. The resulting scaffold contains 75 wt% β-TCP powder and 25 wt% polycaprolactone.

[0182] The ink is also converted into pellets (eg, the filaments are formed into small pellets) and fed from a hopper to a mini-screw extrusion head, which melts the material and extrudes it through fine nozzles.

[0183] The ink is then freeze-milled into a fine powder, which is then heated with a laser using the SLS process to form the desired configuration.

[0184] [Table 11]

[0185] Ink Formulation #9 This 3D printing ink material is a flexible polymer-ceramic composite containing β-TCP, which can be tethered to therapeutic agents such as tBMP2. The formulation contains two sacrificial pore-forming agents (water-soluble polyethylene glycol and water-soluble glucose) to expose a larger β-TCP surface area for drug binding. This ink is a medium-viscosity formulation that can be molded into 1.75 mm filaments for 3D printing with a RepRap-style FFF 3D printer (e.g., the Prusa i3 MK3S 3D Printer) equipped with a 400 μm diameter nozzle. Blends of higher molecular weight polyethylene glycols (8000 MW and 20,000 MW for FFF 3D printing, 1500 MW for syringe-based bioprinting) result in a more viscous material that aids in the extrusion of 1.75 mm diameter filaments. After 3D printing, the resulting scaffold is immersed in water to dissolve the sacrificial polyethylene glycol and sucrose pore-forming agent. The resulting scaffold contains 75% by weight of β-TCP powder and 25% by weight of polycaprolactone.

[0186] The ink is also converted into pellets (eg, the filaments are formed into small pellets) and fed from a hopper to a mini-screw extrusion head, which melts the material and extrudes it through fine nozzles.

[0187] The ink is then freeze-milled into a fine powder, which is then heated with a laser using the SLS process to form the desired configuration.

[0188] [Table 12]

[0189] Ink Formulation #10 This 3D printing ink material is a flexible polymer-ceramic composite containing β-TCP, which can be tethered to therapeutic agents such as tBMP2. The formulation contains a sacrificial pore-forming agent (water-soluble polyethylene glycol) to expose a larger β-TCP surface area for drug binding. The formulation utilizes a 95 mol% caprolactone-5 mol% glycolide copolymer for faster bioabsorption compared to polycaprolactone. This ink is a medium-viscosity formulation that can be molded into 1.75 mm filaments for 3D printing with a RepRap-style FFF 3D printer (e.g., the Prusa i3 MK3S 3D printer) equipped with a 400 μm diameter nozzle. Blends of higher molecular weight polyethylene glycols (8000 MW and 20,000 MW for FFF 3D printing, 1500 MW for syringe-based bioprinting) result in a more viscous material that aids in the extrusion of 1.75 mm diameter filaments. After 3D printing, the resulting scaffold was immersed in water to dissolve the sacrificial polyethylene glycol pore former. The resulting scaffold contained 75% by weight of β-TCP powder and 25% by weight of caprolactone / glycolide copolymer (95:5).

[0190] The ink is also converted into pellets (eg, the filaments are formed into small pellets) and fed from a hopper to a mini-screw extrusion head, which melts the material and extrudes it through fine nozzles.

[0191] The ink is then freeze-milled into a fine powder, which is then heated with a laser using the SLS process to form the desired configuration.

[0192] [Table 13]

[0193] Ink Formulation #11 This 3D printing ink material is a flexible polymer-ceramic composite containing β-TCP, which can be tethered to therapeutic agents such as tBMP2. The formulation contains a sacrificial pore-forming agent (water-soluble polyethylene glycol) to expose a larger β-TCP surface area for drug binding. The formulation utilizes a 90 mol% caprolactone-10 mol% glycolide copolymer for faster bioresorption compared to polycaprolactone. This ink is a medium-viscosity formulation that can be molded into 1.75 mm filaments for 3D printing with a RepRap-style FFF 3D printer (e.g., the Prusa i3 MK3S 3D printer) equipped with a 400 μm diameter nozzle. Blends of higher molecular weight polyethylene glycols (8000 MW and 20,000 MW for FFF 3D printing, 1500 MW for syringe-based bioprinting) result in a more viscous material that aids in the extrusion of 1.75 mm diameter filaments. After 3D printing, the resulting scaffold was immersed in water to dissolve the sacrificial polyethylene glycol pore former. The resulting scaffold contained 75 wt% β-TCP powder and 25 wt% caprolactone / glycolide copolymer (90:10).

[0194] The ink is also converted into pellets (eg, the filaments are formed into small pellets) and fed from a hopper to a mini-screw extrusion head, which melts the material and extrudes it through fine nozzles.

[0195] The ink is then freeze-milled into a fine powder, which is then heated with a laser using the SLS process to form the desired configuration.

[0196] [Table 14]

[0197] Ink Formulation #12 This 3D printing ink material is a flexible polymer-ceramic composite containing β-TCP, which can be tethered to therapeutic agents such as tBMP2. The formulation contains a sacrificial pore-forming agent (water-soluble polyethylene glycol) to expose a larger β-TCP surface area for drug binding. The formulation utilizes 50 mol%:50 mol% poly(D,L-lactide-co-glycolide) copolymer for its fast bioresorption properties compared to polycaprolactone. This ink is a medium-viscosity formulation that can be molded into 1.75 mm filaments for 3D printing with a RepRap-style FFF 3D printer (e.g., Prusa i3 MK3S 3D printer) equipped with a 400 μm diameter nozzle. Blends of higher molecular weight polyethylene glycols (8000 MW and 20000 MW for FFF 3D printing, 1500 MW for syringe-based bioprinting) result in a more viscous material that aids in the extrusion of 1.75 mm diameter filaments. After 3D printing, the resulting scaffold was immersed in water to dissolve the sacrificial polyethylene glycol pore former. The resulting scaffold contained 75 wt% β-TCP powder and 25 wt% poly(D,L-lactide-co-glycolide) copolymer (50:50).

[0198] The ink is also converted into pellets (eg, the filaments are formed into small pellets) and fed from a hopper to a mini-screw extrusion head, which melts the material and extrudes it through fine nozzles.

[0199] The ink is then freeze-milled into a fine powder, which is then heated with a laser using the SLS process to form the desired configuration.

[0200] [Table 15]

[0201] Ink Formulation #13 This 3D printing ink material is a flexible polymer-ceramic composite containing β-TCP, which can be tethered to a therapeutic agent such as tBMP2. The formulation contains a sacrificial pore-forming agent (water-soluble polyethylene glycol) to expose a larger β-TCP surface area for drug binding. 3D printing is performed using the formulation in syringe ink and filament form. After 3D printing, the resulting scaffold is immersed in water to dissolve the sacrificial polyethylene glycol pore-forming agent. The resulting scaffold contains β-TCP and PDS.

[0202] The ink is also converted into pellets (eg, the filaments are formed into small pellets) and fed from a hopper to a mini-screw extrusion head, which melts the material and extrudes it through fine nozzles.

[0203] The ink is then freeze-milled into a fine powder, which is then heated with a laser using the SLS process to form the desired configuration.

[0204] [Table 16]

[0205] Ink Formulation #14 This 3D printing ink material is a flexible polymer-ceramic composite containing β-TCP, which can be tethered to a therapeutic agent such as tBMP2. The formulation contains a sacrificial pore-forming agent (water-soluble polyethylene glycol) to expose a larger β-TCP surface area for drug binding. 3D printing is performed using the formulation in syringe ink and filament form. After 3D printing, the resulting scaffold is immersed in water to dissolve the sacrificial polyethylene glycol pore-forming agent. The resulting scaffold contains β-TCP and PDS-glycolide copolymer (90:10).

[0206] The ink is also converted into pellets (eg, the filaments are formed into small pellets) and fed from a hopper to a mini-screw extrusion head, which melts the material and extrudes it through fine nozzles.

[0207] The ink is then freeze-milled into a fine powder, which is then heated with a laser using the SLS process to form the desired configuration.

[0208] [Table 17]

[0209] Ink Formulation #15 This 3D printing ink material is a flexible polymer-ceramic composite containing β-TCP, which can be tethered to a therapeutic agent such as tBMP2. The formulation contains a sacrificial pore-forming agent (water-soluble polyethylene glycol) to expose a larger β-TCP surface area for drug binding. 3D printing is performed using the formulation in syringe ink and filament form. After 3D printing, the resulting scaffold is immersed in water to dissolve the sacrificial polyethylene glycol pore-forming agent. The resulting scaffold contains a β-TCP and -L-lactide copolymer (90:10).

[0210] The ink is also converted into pellets (eg, the filaments are formed into small pellets) and fed from a hopper to a mini-screw extrusion head, which melts the material and extrudes it through fine nozzles.

[0211] The ink is then freeze-milled into a fine powder, which is then heated with a laser using the SLS process to form the desired configuration.

[0212] [Table 18]

[0213] Ink Formulation #16 This 3D printing ink material is a flexible polymer-ceramic composite containing β-TCP, which can be tethered to therapeutic agents such as tBMP2. This formulation contains a sacrificial pore-forming agent (a water-soluble polyethylene glycol component) to expose a larger β-TCP surface area for drug binding. This ink is a medium-viscosity formulation that can be molded into pellets approximately 3-4 mm in diameter for use as a feedstock in a thermofused granule fabrication (FGF) 3D printer (e.g., Piocreat G5) equipped with a 300-1,000 μm diameter nozzle. After 3D printing, the resulting scaffold is immersed in water to dissolve the sacrificial polyethylene glycol pore-forming agent. Blends of higher molecular weight polyethylene glycols (8000 MW and 35,000 MW for FGF 3D printing, 1500 MW for syringe-based bioprinting) result in a more viscous material. After 3D printing, the resulting scaffold is immersed in water to dissolve the sacrificial polyethylene glycol pore-forming agent. The resulting scaffold contains 75% by weight of β-TCP powder and 25% by weight of polycaprolactone. Using this ink, two exemplary scaffolds were prepared by FGF 3D printing. Images of the scaffolds are shown in Figures 13A-13D and 14A-14D.

[0214] The ink is prepared in pellet or filament form for FGF or FFF printing, respectively. The ink is cryo-milled to a fine powder, for example for SLS printing.

[0215] [Table 19]

[0216] Ink Formulation #17 This 3D printing ink material is a flexible polymer-ceramic composite containing β-TCP, which can be tethered to therapeutic agents such as tBMP2. This formulation contains a sacrificial pore-forming agent (a water-soluble polyethylene glycol component) to expose a larger β-TCP surface area for drug binding. This ink is a medium-viscosity formulation that can be molded into pellets approximately 3-4 mm in diameter for use as a feedstock in a thermofused granule fabrication (FGF) 3D printer (e.g., Piocreat G5) equipped with a 300-1,000 μm diameter nozzle. After 3D printing, the resulting scaffold is immersed in water to dissolve the sacrificial polyethylene glycol pore-forming agent. Blends of higher molecular weight polyethylene glycols (8000 MW and 35,000 MW for FGF 3D printing, 1500 MW for syringe-based bioprinting) result in a more viscous material. After 3D printing, the resulting scaffold is immersed in water to dissolve the sacrificial PEG and sucrose. The resulting scaffolds contain 50-88 wt% β-TCP powder and 13-50 wt% polycaprolactone.

[0217] The ink is prepared in pellet or filament form for FGF or FFF printing, respectively. The ink is cryo-milled to a fine powder, for example for SLS printing.

[0218] [Table 20]

[0219] Ink Formulation #18 This 3D printing ink material is a flexible polymer-ceramic composite containing β-TCP, which can be tethered to therapeutic agents such as tBMP2. This formulation contains a sacrificial pore-forming agent (a water-soluble polyethylene glycol component) to expose a larger β-TCP surface area for drug binding. This ink is a medium-viscosity formulation that can be molded into pellets approximately 2-4 mm in diameter for use as a feedstock in a thermofused granule fabrication (FGF) 3D printer (e.g., Piocreat G5) equipped with a 300-1,000 μm diameter nozzle. After 3D printing, the resulting scaffold is immersed in water to dissolve the sacrificial polyethylene glycol pore-forming agent. Blends of higher molecular weight polyethylene glycols (8000 MW and 35,000 MW for FGF 3D printing, 1500 MW for syringe-based bioprinting) result in a more viscous material. After 3D printing, the resulting scaffold is immersed in water to dissolve the sacrificial PEG. The resulting scaffolds contain 55–88 wt% β-TCP powder and 13–50 wt% caprolactone / glycolide copolymer (95:5).

[0220] The ink is prepared in pellet or filament form for FGF or FFF printing, respectively. The ink is cryo-milled to a fine powder, for example for SLS printing.

[0221] [Table 21]

[0222] [Table 22]

[0223] [Table 23]

[0224] [Table 24]

[0225] Ink Formulation #19 This 3D printing ink material is a flexible polymer-ceramic composite containing β-TCP, which can be tethered to therapeutic agents such as tBMP2. This formulation contains a sacrificial pore-forming agent (a water-soluble polyethylene glycol component) to expose a larger β-TCP surface area for drug binding. This ink is a medium-viscosity formulation that can be molded into pellets approximately 2-4 mm in diameter for use as a feedstock in a thermofused granule fabrication (FGF) 3D printer (e.g., Piocreat G5) equipped with a 300-1,000 μm diameter nozzle. After 3D printing, the resulting scaffold is immersed in water to dissolve the sacrificial polyethylene glycol pore-forming agent. Blends of higher molecular weight polyethylene glycols (8000 MW and 35,000 MW for FGF 3D printing, 1500 MW for syringe-based bioprinting) result in a more viscous material. After 3D printing, the resulting scaffold is immersed in water to dissolve the sacrificial PEG. The resulting scaffolds contain 55–88 wt% β-TCP powder and 13–50 wt% caprolactone / glycolide copolymer (90:10).

[0226] The ink is prepared in pellet or filament form for FGF or FFF printing, respectively. The ink is cryo-milled to a fine powder, for example for SLS printing.

[0227] [Table 25]

[0228] [Table 26]

[0229] [Table 27]

[0230] [Table 28]

[0231] Ink Formulation #20 This 3D printing ink material is a flexible polymer-ceramic composite containing β-TCP, which can be tethered to therapeutic agents such as tBMP2. This formulation also contains a sacrificial pore-forming agent (a water-soluble polyethylene glycol component) to expose a larger β-TCP surface area for drug binding. This ink is a medium-viscosity formulation that can be molded into pellets approximately 3-4 mm in diameter for use as a feedstock in a thermofused granule fabrication (FGF) 3D printer (e.g., Piocreat G5) equipped with a 300-1,000 μm diameter nozzle. After 3D printing, the resulting scaffold is immersed in water to dissolve the sacrificial polyethylene glycol pore-forming agent. Blends of higher molecular weight polyethylene glycols (8000 MW and 35,000 MW for FGF 3D printing, 1500 MW for syringe-based bioprinting) result in a more viscous material. After 3D printing, the resulting scaffold is immersed in water to dissolve the sacrificial PEG. The resulting scaffolds contain 55–88 wt% β-TCP powder and 13–50 wt% poly(D,L-lactide-co-glycolide) copolymer (50:50).

[0232] The ink is prepared in pellet or filament form for FGF or FFF printing, respectively. The ink is cryo-milled to a fine powder, for example for SLS printing.

[0233] [Table 29]

[0234] Ink Formulation #21 This 3D printing ink material is a flexible polymer-ceramic composite containing β-TCP, which can be tethered to therapeutic agents such as tBMP2. This formulation also contains a sacrificial pore-forming agent (a water-soluble polyethylene glycol component) to expose a larger β-TCP surface area for drug binding. This ink is a medium-viscosity formulation that can be molded into pellets approximately 3-4 mm in diameter for use as a feedstock in a thermofused granule fabrication (FGF) 3D printer (e.g., Piocreat G5) equipped with a 300-1,000 μm diameter nozzle. After 3D printing, the resulting scaffold is immersed in water to dissolve the sacrificial polyethylene glycol pore-forming agent. Blends of higher molecular weight polyethylene glycols (8000 MW and 35,000 MW for FGF 3D printing, 1500 MW for syringe-based bioprinting) result in a more viscous material. After 3D printing, the resulting scaffold is immersed in water to dissolve the sacrificial PEG. The resulting scaffolds contain 55-88 wt% β-TCP powder and 13-50 wt% PDS.

[0235] The ink is prepared in pellet or filament form for FGF or FFF printing, respectively. The ink is cryo-milled to a fine powder, for example for SLS printing.

[0236] [Table 30]

[0237] Ink Formulation #22 This 3D printing ink material is a flexible polymer-ceramic composite containing β-TCP, which can be tethered to therapeutic agents such as tBMP2. This formulation also contains a sacrificial pore-forming agent (a water-soluble polyethylene glycol component) to expose a larger β-TCP surface area for drug binding. This ink is a medium-viscosity formulation that can be molded into pellets approximately 3-4 mm in diameter for use as a feedstock in a thermofused granule fabrication (FGF) 3D printer (e.g., Piocreat G5) equipped with a 300-1,000 μm diameter nozzle. After 3D printing, the resulting scaffold is immersed in water to dissolve the sacrificial polyethylene glycol pore-forming agent. Blends of higher molecular weight polyethylene glycols (8000 MW and 35,000 MW for FGF 3D printing, 1500 MW for syringe-based bioprinting) result in a more viscous material. After 3D printing, the resulting scaffold is immersed in water to dissolve the sacrificial PEG. The resulting scaffolds contain 55–88 wt% β-TCP powder and 13–50 wt% dioxanone / L-lactide copolymer (90:10).

[0238] The ink is prepared in pellet or filament form for FGF or FFF printing, respectively. The ink is cryo-milled to a fine powder, for example for SLS printing.

[0239] [Table 31]

[0240] Ink Formulation #23 This 3D printing ink material is a flexible polymer-ceramic composite containing β-TCP, which can be tethered to therapeutic agents such as tBMP2. This formulation also contains a sacrificial pore-forming agent (a water-soluble polyethylene glycol component) to expose a larger β-TCP surface area for drug binding. This ink is a medium-viscosity formulation that can be molded into pellets approximately 3-4 mm in diameter for use as a feedstock in a thermofused granule fabrication (FGF) 3D printer (e.g., Piocreat G5) equipped with a 300-1,000 μm diameter nozzle. After 3D printing, the resulting scaffold is immersed in water to dissolve the sacrificial polyethylene glycol pore-forming agent. Blends of higher molecular weight polyethylene glycols (8000 MW and 35,000 MW for FGF 3D printing, 1500 MW for syringe-based bioprinting) result in a more viscous material. After 3D printing, the resulting scaffold is immersed in water to dissolve the sacrificial PEG. The resulting scaffolds contain 55-88 wt% β-TCP powder and 13-50 wt% glycolide / L-lactide copolymer (95:5).

[0241] The ink is prepared in pellet or filament form for FGF or FFF printing, respectively. The ink is cryo-milled to a fine powder, for example for SLS printing.

[0242] [Table 32]

[0243] Ink Formulation #24 This 3D printing ink material is a flexible polymer-ceramic composite containing β-TCP, which can be tethered to therapeutic agents such as tBMP2. This formulation also contains a sacrificial pore-forming agent (a water-soluble polyethylene glycol component) to expose a larger β-TCP surface area for drug binding. This ink is a medium-viscosity formulation that can be molded into pellets approximately 3-4 mm in diameter for use as a feedstock in a thermofused granule fabrication (FGF) 3D printer (e.g., Piocreat G5) equipped with a 300-1,000 μm diameter nozzle. After 3D printing, the resulting scaffold is immersed in water to dissolve the sacrificial polyethylene glycol pore-forming agent. Blends of higher molecular weight polyethylene glycols (8000 MW and 35,000 MW for FGF 3D printing, 1500 MW for syringe-based bioprinting) result in a more viscous material. After 3D printing, the resulting scaffold is immersed in water to dissolve the sacrificial PEG. The resulting scaffolds contain 55-88 wt% β-TCP powder and 13-50 wt% poly-l-lactide.

[0244] The ink is prepared in pellet or filament form for FGF or FFF printing, respectively. The ink is cryo-milled to a fine powder, for example for SLS printing.

[0245] [Table 33]

[0246] Example 2: Ink preparation and scaffold fabrication by 3D printing Ink formulation and scaffold #1 Method: To make a 5.3 cc batch of ink, 5.6 g of β-TCP powder, 1.87 g of polycaprolactone powder, 1.87 g of polyethylene glycol flakes, and 0.49 g of sucrose were added to a glass mixing vessel. The vial was placed in a double asymmetric centrifugal mixer (FlackTek Speedmixer) and mixed at low intensity (300 rpm) for 2 minutes to homogenize the powder blend, followed by mixing at high rpm. The mixer was then mixed at high intensity (3500 rpm) for 5 minutes. During mixing, internal friction melted the polycaprolactone and polyethylene glycol, transforming the ink into a viscous molten liquid. This liquid-phase mixing promoted the intimate dispersion of the β-TCP and sucrose powders into the molten polymer blend. The blended ink was allowed to cool for 10–15 minutes and then mixed at 3500 rpm for an additional 5 minutes. The mixing / cooling process was repeated for a total of four 5-minute periods at 3500 rpm. After a fourth mixing at 3500 rpm, the ink charge was poured onto a glass plate and formed into a cylindrical shape approximately 1 cm in diameter and 6 cm long using two spatulas. While the ink was still semi-molten, it was cut into pieces approximately 1-2 cm long using a straight razor.

[0247] 3D printing: The solid polymer / β-TCP pieces were transferred to a 5cc stainless steel syringe (for use with the Allevi 3 Bioprinter). The print head of the Extruder CORE was heated to 135°C and allowed to dwell for approximately 30 minutes to ensure the ink was melted. The ink was printed using a 400 micron inner diameter conical metal Luer-lock tip at 70 psi pressure and a nozzle speed of 7 mm / sec. Scaffolds were 3D printed onto painter's tape attached to a smooth glass or polymer surface, such as a glass microscope slide, a larger glass plate, or the lid of a 96-well plate.

[0248] Post-processing: The 3D printed constructs were soaked in distilled water overnight to dissolve the polyethylene glycol and sucrose from the printed material, resulting in a porous and flexible β-TCP / polycaprolactone composite. The scaffolds were then dried for at least 12 hours to ensure that any residual moisture had evaporated from the porous scaffold before conjugation with therapeutic agents such as tBMP2 protein. The scaffolds were sterilized by immersion in a 70% ethanol solution for 2-4 hours and then allowed to dry for approximately 12 hours in a biosafety cabinet. Images of the scaffolds are shown in Figure 1A-C.

[0249] 3D printing is also performed using an FFF 3D printer. Ink #1 is extruded into a filament, which is then loaded into the FFF 3D printer to produce the 3D printed scaffold. The scaffold is then processed using the post-processing methods outlined above.

[0250] Ink formulation and scaffold #2 Method: To make a 5 cc batch of ink, 5.6 g of β-TCP powder, 1.87 g of 95:5 caprolactone / glycolide copolymer pellets, and 1.87 g of polyethylene glycol flakes were added to a glass mixing vessel. The vial was placed in a double asymmetric centrifugal mixer (FlackTek Speedmixer) and mixed at low intensity (300 rpm) for 2 minutes to homogenize the powder blend, followed by mixing at high rpm. The mixture was mixed at high intensity (3500 rpm) for 5 minutes. During mixing, internal friction melted the 95:5 caprolactone / glycolide copolymer and polyethylene glycol, transforming the ink into a viscous molten liquid. This liquid-phase mixing facilitated intimate dispersion of the β-TCP powder into the molten polymer blend. The blended ink was allowed to cool for 10–15 minutes and then mixed at 3500 rpm for an additional 5 minutes. The mixing / cooling process was repeated for a total of four 5-minute periods at 3500 rpm. After a fourth mixing at 3500 rpm, the ink charge was poured onto a glass plate and formed into a cylindrical shape approximately 1 cm in diameter and 6 cm long using two spatulas. While the ink was still semi-molten, it was cut into pieces approximately 1-2 cm long using a straight razor.

[0251] 3D printing: The solid polymer / β-TCP pieces were transferred to a 5cc stainless steel syringe (for use with the Allevi 3 Bioprinter). The print head of the Extruder CORE was heated to 130°C and allowed to dwell for approximately 30 minutes to ensure the ink was melted. The ink was printed using a conical metal Luer-lock tip with an inner diameter of 320 microns at 80 psi pressure and a nozzle speed of 6 mm / sec. Scaffolds were 3D printed onto painter's tape attached to a smooth glass or polymer surface, such as a glass microscope slide, a larger glass plate, or the lid of a 96-well plate.

[0252] Post-processing: The 3D printed constructs were soaked in distilled water overnight to dissolve the polyethylene glycol from the printed material, resulting in a porous and flexible β-TCP / 95:5 caprolactone / glycolide copolymer composite. The scaffolds were allowed to dry for at least 12 hours to ensure that any residual moisture had evaporated from the porous scaffold before conjugation with therapeutic agents such as tBMP2. The scaffolds were sterilized by immersion in a 70% ethanol solution for 2-4 hours and then allowed to dry for approximately 12 hours in a biosafety cabinet. Images of the scaffolds are shown in Figure 2A-C.

[0253] 3D printing was also performed using an FFF 3D printer. Ink #2 was extruded into a filament, which was then loaded into the FFF 3D printer to produce the 3D printed scaffold. The scaffold was then processed using the post-processing methods outlined above.

[0254] Ink formulation and scaffold #3 Method: To make a 5 cc batch of ink, 5.6 g of β-TCP powder, 1.87 g of 90:10 caprolactone / glycolide copolymer flakes, and 1.87 g of polyethylene glycol flakes were added to a glass mixing vessel. The mixture was placed in a glass bottle in a double asymmetric centrifugal mixer (FlackTek Speedmixer) and mixed at low intensity (300 rpm) for 2 minutes to homogenize the powder blend, followed by mixing at high rpm. This was then mixed at high intensity (3500 rpm) for 5 minutes. During mixing, internal friction melted the 90:10 caprolactone / glycolide copolymer and polyethylene glycol, transforming the ink into a viscous molten liquid. This liquid-phase mixing facilitated intimate dispersion of the β-TCP powder into the molten polymer blend. The blended ink was allowed to cool for 10–15 minutes and then mixed at 3500 rpm for an additional 5 minutes. The mixing / cooling process was repeated for a total of four 5-minute periods at 3500 rpm. After a fourth mixing at 3500 rpm, the ink charge was poured onto a glass plate and formed into a cylindrical shape approximately 1 cm in diameter and 6 cm long using two spatulas. While the ink was still semi-molten, it was cut into pieces approximately 1-2 cm long using a straight razor.

[0255] 3D printing: The solid polymer / β-TCP pieces were transferred to a 5cc stainless steel syringe (for use with the Allevi 3 Bioprinter). The print head of the Extruder CORE was heated to 130°C and allowed to dwell for approximately 30 minutes to ensure the ink was melted. The ink was printed using a conical metal Luer-lock tip with an inner diameter of 320 microns at 45 psi pressure and a nozzle speed of 7 mm / sec. Scaffolds were 3D printed onto painter's tape attached to a smooth glass or polymer surface, such as a glass microscope slide, a larger glass plate, or the lid of a 96-well plate.

[0256] Post-processing: The 3D printed constructs were soaked in distilled water overnight to dissolve the polyethylene glycol from the printed material, resulting in a porous and flexible β-TCP / 90:10 caprolactone / glycolide copolymer composite. The scaffolds were allowed to dry for at least 12 hours to ensure that any residual moisture had evaporated from the porous scaffold before conjugation with therapeutic agents such as tBMP2. The scaffolds were sterilized by immersion in a 70% ethanol solution for 2-4 hours and then allowed to dry for approximately 12 hours in a biosafety cabinet. Images of the scaffolds are shown in Figure 3A-C.

[0257] 3D printing was also performed using an FFF 3D printer. Ink #3 was extruded into filament, which was then loaded into the FFF 3D printer to produce the 3D printed scaffold. The scaffold was then processed using the post-processing methods outlined above.

[0258] Ink formulation and scaffold #4 Method: To prepare a 2.5 cc batch of ink, 2.8 g of β-TCP powder, 0.94 g of 50:50 poly(D,L-lactide-co-glycolide) copolymer chunks, and 0.94 g of polyethylene glycol flakes were added to a glass mixing vessel. The vial was placed in a double asymmetric centrifugal mixer (FlackTek Speedmixer) and mixed at low intensity (300 rpm) for 2 minutes to homogenize the powder blend, followed by mixing at high intensity (3500 rpm) for 5 minutes. During mixing, internal friction caused the 50:50 poly(D,L-lactide-co-glycolide) copolymer and polyethylene glycol to flow, transforming the ink into a viscous molten liquid. This liquid-phase mixing facilitated intimate dispersion of the β-TCP powder into the molten polymer blend. The blended ink was allowed to cool for 10–15 minutes and then mixed at 3500 rpm for an additional 5 minutes. The mixing / cooling process was repeated for a total of four 5-minute mixing runs at 3500 rpm. After the fourth run at 3500 rpm, the ink charge was poured onto a glass plate and formed into a roughly 1 cm diameter x 3 cm long cylinder using two spatulas. While the ink was still semi-molten, it was cut into approximately 1-2 cm long pieces using a straight razor.

[0259] 3D printing: The solid polymer / β-TCP pieces were transferred to a 5cc stainless steel syringe (for use with the Allevi 3 Bioprinter). The print head of the Extruder CORE was heated to 85°C and allowed to dwell for approximately 30 minutes to ensure the ink was melted. The ink was printed using a 400 micron inner diameter conical metal Luer-lock tip at 60 psi pressure and a nozzle speed of 7 mm / sec. Scaffolds were 3D printed onto painter's tape attached to a smooth glass or polymer surface, such as a glass microscope slide, a larger glass plate, or the lid of a 96-well plate.

[0260] Post-processing: The 3D printed construct was soaked in distilled water overnight to dissolve the polyethylene glycol from the printed material, resulting in a porous and flexible β-TCP / 50:50 poly(D,L-lactide-co-glycolide) composite. The scaffold was allowed to dry for at least 12 hours to ensure that any residual moisture had evaporated from the porous scaffold before conjugation with a therapeutic agent such as tBMP2. The scaffold was sterilized by immersion in a 70% ethanol solution for 2-4 hours and then allowed to dry for approximately 12 hours in a biosafety cabinet. Images of the scaffold are shown in Figure 4A-4C.

[0261] 3D printing was also performed using an FFF 3D printer. Ink #4 was extruded into a filament, which was then loaded into the FFF 3D printer to produce the 3D printed scaffold. The scaffold was then processed using the post-processing methods outlined above.

[0262] Ink formulation and scaffold #5 Method: To prepare a 5 cc batch of ink, 5.6 g of β-TCP powder, 1.87 g of polycaprolactone powder, and 1.87 g of polyethylene glycol flakes were added to a glass mixing vessel. The glass vessel was placed in a double asymmetric centrifugal mixer (FlackTek Speedmixer) and mixed at low intensity (300 rpm) for 2 minutes to homogenize the powder blend, followed by mixing at high rpm. This was then mixed at high intensity (3500 rpm) for 5 minutes. During mixing, internal friction melted the polycaprolactone and polyethylene glycol, transforming the ink into a viscous molten liquid. This liquid-phase mixing facilitated the intimate dispersion of the β-TCP powder into the molten polymer blend. The molten ink was poured onto a glass plate to cool and smoothed with a spatula into a layer approximately 3 mm thick. After cooling, it was cut into approximately 3-4 mm pellets using scissors. This process was repeated for two additional 5 cc batches, producing a total of 15 cc of pellets for filament extrusion.

[0263] Filament Preparation: 15 cc of 3-4 mm pellets were loaded into the hopper of a filament extruder (Filabot EX2 Filament Extruder). A three-times-long extended melt filter nozzle (filter screen removed) with a 1.75 mm diameter hole was used. A cooling fan was positioned near the extrusion nozzle to accelerate solidification of the extruded filament. 1.75 mm diameter filament was extruded at an extrusion temperature of 62°C and an extrusion speed of 1 / 2 on the analog dial (approximately 1 cm / sec extrusion speed).

[0264] 3D printing: 1.75 mm filament was loaded into a Prusa i3 MK3S 3D printer. The filament material was printed using a 400 micron brass nozzle, an extruder temperature of 105°C, and a print speed of 15 mm / s.

[0265] Post-processing: The 3D printed constructs were soaked in distilled water overnight to dissolve the polyethylene glycol from the printed material, resulting in a porous and flexible β-TCP / polycaprolactone composite. The scaffolds were allowed to dry for at least 12 hours to ensure that any residual moisture had evaporated from the porous scaffold before conjugation with therapeutic agents such as tBMP2. The scaffolds were sterilized by immersion in a 70% ethanol solution for 2-4 hours and then allowed to dry for approximately 12 hours in a biosafety cabinet. Images of the scaffolds are shown in Figure 5A-C.

[0266] Ink Formulation and Scaffold #6 Methods: To make a 5.5 cc batch of ink, 5.6 g of β-TCP powder, 1.87 g of polycaprolactone powder, 1.87 g of polyethylene glycol flakes, and 1.04 g of sodium bicarbonate were added to a glass mixing vessel. The vial was placed in a double asymmetric centrifugal mixer (FlackTek Speedmixer) and mixed at low intensity (300 rpm) for 2 minutes to homogenize the powder blend, followed by mixing at high intensity (3500 rpm) for 2 minutes. During mixing, internal friction melted the polycaprolactone and polyethylene glycol, transforming the ink into a viscous molten liquid. This liquid-phase mixing facilitated the intimate dispersion of the β-TCP and sodium bicarbonate powders into the molten polymer blend. The molten ink was poured onto a glass plate to cool and smoothed with a spatula into a layer approximately 3 mm thick. After cooling, it was cut into approximately 3-4 mm pellets using scissors. This was repeated for two additional 5.5 cc batches to produce a total of 16.5 cc of pellets for filament extrusion.

[0267] Filament Preparation: 16.5 cc of 3-4 mm pellets were loaded into the hopper of a filament extruder (Filabot EX2 Filament Extruder). A three-times-long extended melt filter nozzle (filter screen removed) with a 1.75 mm diameter hole was used. A cooling fan was positioned near the extrusion nozzle to accelerate solidification of the extruded filament. 1.75 mm diameter filament was extruded at an extrusion temperature of 62°C and an extrusion speed of 1 / 2 on the analog dial (approximately 1 cm / sec extrusion rate).

[0268] 3D printing: 1.75 mm of filament was loaded into a Prusa i3 MK3S 3D printer. The filament material was printed using a 400 micron brass nozzle, an extruder temperature of 155°C, and a print speed of 15 mm / s.

[0269] Post-processing: The 3D printed construct was soaked in distilled water overnight to dissolve the polyethylene glycol and sodium bicarbonate (a by-product of sodium bicarbonate thermal decomposition) from the foam-like printed material, resulting in a porous and flexible β-TCP / polycaprolactone composite. The scaffold was then dried for at least 12 hours to ensure that any residual moisture had evaporated from the porous scaffold before conjugation with a therapeutic agent such as tBMP2. The scaffold was then sterilized by immersion in a 70% ethanol solution for 2-4 hours and allowed to dry for approximately 12 hours in a biosafety cabinet. Images of the scaffold are shown in Figure 6A-6C.

[0270] Ink Formulation and Scaffold #7 Method: To make a 10 cc batch of ink, 11.2 g of β-TCP powder, 3.74 g of dioxanone / L-lactide flakes, and 3.74 g of polyethylene glycol flakes were added to a glass mixing vessel. The vial was placed in a double asymmetric centrifugal mixer (FlackTek Speedmixer) and mixed at low intensity (300 rpm) for 2 minutes to homogenize the powder blend, followed by mixing at high rpm. The vial was then transferred to a hot plate and heated until it reached 185°C (as measured by an infrared thermometer). The vial was then immediately returned to the double asymmetric centrifugal mixer and mixed at high intensity (3500 rpm) for 5 minutes. Liquid-phase mixing promotes intimate dispersion of the β-TCP powder into the molten polymer blend. The vial was then returned to the hot plate and the temperature was increased to 185°C. Once this temperature was reached, the vial was immediately returned to the double asymmetric centrifugal mixer and mixed at 3500 rpm for an additional 5 minutes. The mixing / heating process was repeated for a total of four 5-minute mixing times at 3500 rpm. After the fourth mixing at 3500 rpm, the ink charge was poured onto a glass plate and formed into a cylindrical shape approximately 1 cm in diameter and 6 cm long using two spatulas. While the ink was still semi-molten, it was cut into approximately 1-2 cm long pieces using a straight razor.

[0271] 3D printing: The solid polymer / β-TCP pieces were transferred to a 5cc stainless steel syringe (for use with the Allevi 3 Bioprinter). The print head of the Extruder CORE was heated to 110°C and allowed to dwell for approximately 30 minutes to ensure the ink was melted. The ink was printed using a 400 micron inner diameter conical metal Luer-lock tip at 15 psi pressure and a nozzle speed of 10 mm / sec. Scaffolds were 3D printed onto painter's tape attached to a smooth glass or polymer surface, such as a glass microscope slide, a larger glass plate, or the lid of a 96-well plate.

[0272] Post-processing: The 3D printed constructs were soaked in distilled water overnight to dissolve the polyethylene glycol from the printed material. This process produced a porous and flexible β-TCP / 90:10 dioxanone-L-lactide copolymer composite. The scaffolds were allowed to dry for at least 12 hours to ensure that any residual moisture had evaporated from the porous scaffold before conjugation with a therapeutic agent, such as tBMP2 protein. The scaffolds were sterilized by immersion in a 70% ethanol solution for 2-4 hours and then allowed to dry for approximately 12 hours in a biosafety cabinet. Images of the scaffolds are shown in Figures 7A-7C.

[0273] 3D printing was also performed using an FFF 3D printer. Ink #7 was extruded into filament, which was then loaded into an FFF 3D printer to produce the 3D printed scaffold. The scaffold was then processed using the post-processing methods outlined above.

[0274] Ink Formulation and Scaffold #8 Method: To make a 16 cc batch of ink, 18 g of β-TCP powder, 6 g of polycaprolactone powder, 3 g of polyethylene glycol (8,000 MW) flakes, and 3 g of polyethylene glycol (20,000 MW) flakes were added to a Teflon mixing vessel. The Teflon vessel was placed in a double asymmetric centrifugal mixer (FlackTek Speedmixer) and mixed at low intensity (300 rpm) for 2 minutes to homogenize the powder blend, followed by mixing at high rpm. The Teflon vessel was then mixed at high intensity (3500 rpm) for 2.5 minutes. During mixing, internal friction melted the polycaprolactone and polyethylene glycol, transforming the ink into a viscous molten liquid. This liquid-phase mixing facilitated intimate dispersion of the β-TCP powder into the molten polymer blend. The molten ink was poured onto a glass plate to cool and then spread with a spatula to form a layer approximately 3 mm thick. This was allowed to cool for 10-15 minutes. The mixture was returned to the Teflon container and mixed at high intensity (3500 rpm) for an additional 2.5 minutes. The mixing / cooling process was repeated for a total of four 2.5 minute mixes at 3500 rpm. After the fourth and final mix, the ink was allowed to cool and then cut into approximately 3-4 mm pellets using scissors.

[0275] Filament Preparation: 16 cc of 3-4 mm pellets were loaded into the hopper of a filament extruder (Filabot EX2 Filament Extruder). A three-times-long extended melt filter nozzle (filter screen removed) with a 1.75 mm diameter hole was used. A cooling fan was positioned near the extrusion nozzle to accelerate solidification of the extruded filament. 1.75 mm diameter filament was extruded at an extrusion temperature of 62°C and an extrusion speed of 1 / 2 on the analog dial (approximately 1 cm / sec extrusion rate).

[0276] 3D printing: 1.75 mm filament was loaded into a Prusa i3 MK3S 3D printer. The filament material was printed using a 400 micron brass nozzle, an extruder temperature of 105°C, and a print speed of 15 mm / s.

[0277] Post-processing: The 3D printed constructs were soaked in distilled water overnight to dissolve the polyethylene glycol from the printed material, resulting in a porous and flexible β-TCP / polycaprolactone composite. The scaffolds were allowed to dry for at least 12 hours to ensure that any residual moisture had evaporated from the porous scaffold before conjugation with therapeutic agents such as tBMP2. The scaffolds were sterilized by immersion in a 70% ethanol solution for 2-4 hours and then allowed to dry for approximately 12 hours in a biosafety cabinet. Images of the scaffolds are shown in Figure 8A-C.

[0278] Ink Formulation and Scaffold #9 Method: To make a 16.3 cc batch of ink, 17.1 g of β-TCP powder, 5.7 g of polycaprolactone powder, 2.85 g of polyethylene glycol (8,000 MW) flakes, 2.85 g of polyethylene glycol (20,000 MW) flakes, and 1.5 g of sucrose were added to a Teflon mixing vessel. The Teflon vessel was placed in a double asymmetric centrifugal mixer (FlackTek Speedmixer) and mixed at low intensity (300 rpm) for 2 minutes to homogenize the powder blend, followed by mixing at high rpm. The Teflon vessel was then mixed at high intensity (3500 rpm) for 2 minutes. During mixing, internal friction melted the polycaprolactone and polyethylene glycol, transforming the ink into a viscous molten liquid. This liquid-phase mixing facilitated intimate dispersion of the β-TCP powder into the molten polymer blend. The molten ink was poured onto a glass plate to cool and smoothed with a spatula into a layer approximately 3 mm thick. This was allowed to cool for 10-15 minutes. The mixture was returned to the Teflon container and mixed at high intensity (3500 rpm) for an additional 2 minutes. The mixing / cooling process was repeated for a total of four 2-minute mixes at 3500 rpm. After the ink had cooled after the fourth and final mix, it was cut into approximately 3-4 mm pellets using scissors.

[0279] Filament Preparation: 16.3 cc of 3-4 mm pellets were loaded into the hopper of a filament extruder (Filabot EX2 Filament Extruder). A three-times-long extended melt filter nozzle (filter screen removed) with a 1.75 mm diameter hole was used. A cooling fan was positioned near the extrusion nozzle to accelerate solidification of the extruded filament. 1.75 mm diameter filament was extruded at an extrusion temperature of 62°C and an extrusion speed of 1 / 2 on the analog dial (approximately 1 cm / sec extrusion rate).

[0280] 3D printing: 1.75 mm of filament was loaded into a Prusa i3 MK3S 3D printer. The filament material was printed using a 400 micron brass nozzle, an extruder temperature of 140°C, and a print speed of 10 mm / s.

[0281] Post-processing: The 3D printed constructs were soaked in distilled water overnight to dissolve the polyethylene glycol and sucrose from the printed material, resulting in a porous and flexible β-TCP / polycaprolactone composite. The scaffolds were allowed to dry for at least 12 hours to ensure that any residual moisture had evaporated from the porous scaffold before conjugation with therapeutic agents such as tBMP2. The scaffolds were sterilized by immersion in a 70% ethanol solution for 2-4 hours and then allowed to dry for approximately 12 hours in a biosafety cabinet. Images of the scaffolds are shown in Figure 9A-C.

[0282] Ink Formulation and Scaffold #10 Method: To prepare a 16 cc batch of ink, 18 g of β-TCP powder, 6 g of caprolactone / glycolide copolymer (95:5) pellets, 3 g of polyethylene glycol (8,000 MW) flakes, and 3 g of polyethylene glycol (20,000 MW) flakes were added to a Teflon mixing vessel. The Teflon vessel was placed in a double asymmetric centrifugal mixer (FlackTek Speedmixer) and mixed at low intensity (300 rpm) for 2 minutes to homogenize the powder blend, followed by mixing at high rpm. The Teflon vessel was then mixed at high intensity (3500 rpm) for 2.5 minutes. During mixing, internal friction melted the caprolactone / glycolide copolymer (95:5) and polyethylene glycol, transforming the ink into a viscous molten liquid. This liquid-phase mixing facilitated intimate dispersion of the β-TCP powder into the molten polymer blend. The molten ink was poured onto a glass plate to cool and smoothed with a spatula into a layer approximately 3 mm thick. This was allowed to cool for 10-15 minutes. The mixture was returned to the Teflon container and mixed at high intensity (3500 rpm) for an additional 4 minutes. The mixing / cooling process was repeated for one 2.5 minute mix and three 4 minute mixes at 3500 rpm. After the fourth and final mix, the ink was cooled and then cut into approximately 3-4 mm pellets using scissors.

[0283] Filament Preparation: 16 cc of 3-4 mm pellets were loaded into the hopper of a filament extruder (Filabot EX2 Filament Extruder). A three-times-long extended melt filter nozzle (filter screen removed) with a 1.75 mm diameter hole was used. A cooling fan was positioned near the extrusion nozzle to accelerate solidification of the extruded filament. 1.75 mm diameter filament was extruded at an extrusion temperature of 62°C and an extrusion speed of 1 / 2 on the analog dial (approximately 1 cm / sec extrusion rate).

[0284] 3D printing: 1.75 mm filament was loaded into a Prusa i3 MK3S 3D printer. The filament material was printed using a 400 micron brass nozzle, an extruder temperature of 150°C, and a print speed of 10 mm / s.

[0285] Post-processing: The 3D printed constructs were soaked in distilled water overnight to dissolve the polyethylene glycol from the printed material, resulting in a porous and flexible β-TCP / caprolactone / glycolide copolymer (95:5) composite. The scaffolds were allowed to dry for at least 12 hours to ensure that any residual moisture had evaporated from the porous scaffold before conjugation with therapeutic agents such as tBMP2. The scaffolds were sterilized by immersion in a 70% ethanol solution for 2-4 hours and then allowed to dry for approximately 12 hours in a biosafety cabinet. Images of the scaffolds are shown in Figure 10A-C.

[0286] Ink Formulation and Scaffold #11 Method: To prepare a 16 cc batch of ink, 18 g of β-TCP powder, 6 g of caprolactone / glycolide copolymer (90:10) flakes, 3 g of polyethylene glycol (8,000 MW) flakes, and 3 g of polyethylene glycol (20,000 MW) flakes were added to a Teflon mixing vessel. The Teflon vessel was placed in a double asymmetric centrifugal mixer (FlackTek Speedmixer) and mixed at low intensity (300 rpm) for 2 minutes to homogenize the powder blend, followed by mixing at high rpm. The Teflon vessel was then mixed at high intensity (3500 rpm) for 2 minutes. During mixing, internal friction melted the caprolactone / glycolide copolymer (90:10) and polyethylene glycol, transforming the ink into a viscous molten liquid. This liquid-phase mixing facilitated intimate dispersion of the β-TCP powder into the molten polymer blend. The molten ink was poured onto a glass plate to cool and smoothed with a spatula into a layer approximately 3 mm thick. This was allowed to cool for 10-15 minutes. The mixture was returned to the Teflon container and mixed at high intensity (3500 rpm) for an additional 5 minutes. The mixing / cooling process was repeated once for 2 minutes at 3500 rpm, and three times for 4 minutes. After the fourth and final mix, the ink was allowed to cool and then cut into approximately 3-4 mm pellets using scissors.

[0287] Filament Preparation: 16 cc of 3-4 mm pellets were loaded into the hopper of a filament extruder (Filabot EX2 Filament Extruder). A three-times-long extended melt filter nozzle (filter screen removed) with a 1.75 mm diameter hole was used. A cooling fan was positioned near the extrusion nozzle to accelerate solidification of the extruded filament. 1.75 mm diameter filament was extruded at an extrusion temperature of 62°C and an extrusion speed of 1 / 2 on the analog dial (approximately 1 cm / sec extrusion rate).

[0288] 3D printing: 1.75 mm of filament was loaded into a Prusa i3 MK3S 3D printer. The filament material was printed using a 400 micron brass nozzle, an extruder temperature of 150°C, a print bed temperature of 40°C, and a print speed of 10 mm / s.

[0289] Post-processing: The 3D printed constructs were soaked in distilled water overnight to dissolve the polyethylene glycol from the printed material, resulting in a porous and flexible β-TCP / caprolactone / glycolide copolymer (90:10) composite. The scaffolds were allowed to dry for at least 12 hours to ensure that any residual moisture had evaporated from the porous scaffold before conjugation with a therapeutic agent such as tBMP2. The scaffolds were sterilized by immersion in a 70% ethanol solution for 2-4 hours and then allowed to dry for approximately 12 hours in a biosafety cabinet. Images of the scaffolds are shown in Figures 11A-C.

[0290] Ink Formulation and Scaffold #12 Method: To make an 8 cc batch of ink, 9 g of β-TCP powder, 3 g of poly(D,L-lactide-co-glycolide) copolymer (50:50) chunks, 1.5 g of polyethylene glycol (8,000 MW) flakes, and 1.5 g of polyethylene glycol (20,000 MW) flakes were added to a Teflon mixing vessel. The Teflon vessel was placed in a double asymmetric centrifugal mixer (FlackTek Speedmixer) and mixed at low intensity (300 rpm) for 2 minutes to homogenize the powder blend, followed by mixing at high rpm. The Teflon vessel was then mixed at high intensity (3500 rpm) for 2 minutes. During mixing, internal friction melted the poly(D,L-lactide-co-glycolide) copolymer (50:50) and polyethylene glycol, transforming the ink into a viscous, molten liquid. This liquid-phase mixing promotes intimate dispersion of the β-TCP powder into the molten polymer blend. The molten ink was poured onto a glass plate to cool and smoothed with a spatula into a layer approximately 3 mm thick. This was allowed to cool for 10-15 minutes. The mixture was returned to the Teflon container and mixed at high intensity (3500 rpm) for an additional 2.5 minutes. The mixing / cooling process was repeated once for 2 minutes at 3500 rpm and twice for 2.5 minutes. After the ink cooled after the third and final mix, it was cut into approximately 3-4 mm pellets using scissors.

[0291] Filament Preparation: 8 cc of 3-4 mm pellets were loaded into the hopper of a filament extruder (Filabot EX2 Filament Extruder). A three-times-long extended melt filter nozzle (filter screen removed) with a 1.75 mm diameter hole was used. A cooling fan was positioned near the extrusion nozzle to accelerate solidification of the extruded filament. 1.75 mm diameter filament was extruded at an extrusion temperature of 62°C and an extrusion speed of 1 / 2 on the analog dial (approximately 1 cm / sec extrusion speed).

[0292] 3D printing: 1.75 mm of filament was loaded into a Prusa i3 MK3S 3D printer. The filament material was printed using a 400 micron brass nozzle, an extruder temperature of 140°C, and a print speed of 10 mm / s.

[0293] Post-processing: The 3D printed constructs were soaked in distilled water overnight to dissolve the polyethylene glycol from the printed material, resulting in a porous and flexible β-TCP / poly(D,L-lactide-co-glycolide) copolymer (50:50) composite. The scaffolds were allowed to dry for at least 12 hours to ensure that any residual moisture had evaporated from the porous scaffold before conjugation with a therapeutic agent such as tBMP2. The scaffolds were sterilized by immersion in a 70% ethanol solution for 2-4 hours and then allowed to dry for approximately 12 hours in a biosafety cabinet. Images of the scaffolds are shown in Figures 12A-C.

[0294] Ink Formulation and Scaffold #13 Ink #13 is printed by syringe-based melt extrusion printing, for example, using the methods described for printing inks #1 through #4. Ink #13 is printed by fused filament fabrication 3D printing, for example, using the methods described for printing inks #5 and #6.

[0295] Ink Formulation and Scaffold #14 Ink #14 is printed by syringe-based melt extrusion printing, for example, using the methods described for printing inks #1 through #4. Ink #14 is printed by fused filament fabrication 3D printing, for example, using the methods described for printing inks #5 and #6.

[0296] Ink Formulation and Scaffold #15 Ink #9 is printed by syringe-based melt extrusion printing, for example, using the methods described for printing Inks #1-4. Ink #15 is printed by fused filament fabrication 3D printing, for example, using the methods described for printing Ink #5 and Ink #6.

[0297] Ink Formulation and Scaffold #16 Method: To prepare a 16 cc batch of ink, 18 g of β-TCP powder, 6 g of polycaprolactone powder, 3 g of polyethylene glycol (8,000 MW) flakes, and 3 g of polyethylene glycol (35,000 MW) flakes were added to a Teflon mixing vessel. The Teflon vessel was placed in a double asymmetric centrifugal mixer (FlackTek Speedmixer) and mixed at low intensity (300 rpm) for 2 minutes to homogenize the powder blend, followed by mixing at high rpm. The powder blend was then mixed at high intensity (3500 rpm) for 2.5 minutes. During mixing, internal friction melted the polycaprolactone and polyethylene glycol, transforming the ink into a viscous molten liquid. This liquid-phase mixing facilitated intimate dispersion of the β-TCP powder into the molten polymer blend. The molten ink was poured onto a glass plate to cool and spread evenly with a spatula to form a layer approximately 3 mm thick. The ink was allowed to cool for 10-15 minutes. After this cooling step, the ink was mixed again for 2.5 minutes at 3500 rpm. This mixing and cooling process (10-15 minutes of cooling, followed by 2.5 minutes of mixing at 3500 rpm) was repeated four times. After the ink had cooled after the fourth and final mixing, scissors were used to cut the ink into feedstock squares of approximately 4 mm to 6 mm for pelletization.

[0298] Pellet production: An 850 μm diameter nozzle was attached to the metal barrel of a heated pneumatic extrusion device (e.g., an Allevi 3 bioprinter equipped with a 5 mL steel syringe barrel). 3–4 cc of 4–6 mm square feedstock was loaded into the barrel. The barrel was heated to a temperature of approximately 80°C to approximately 120°C, e.g., 100°C. After 15 minutes, additional square feedstock was added to the barrel until it was filled and pushed into the molten material with a spatula. After another 10 minutes, the ink was confirmed to be completely melted. The air pressure of the pneumatic extrusion device was then set to approximately 50–100 psi, e.g., 55 psi. A platform was provided below the extrusion tip as a substrate for extruding the pellets. In one example, the platform can be a smooth silicone sheet material. Using manually generated Marlin G-code, up to 120 pellets were extruded in a rectangular array, resulting in approximately equiaxed pellets with diameters of approximately 3–4 mm.

[0299] 3D printing: The pellets were then loaded into the hopper of a Piocreat G5 FGF 3D printer. The material was printed using a nozzle diameter of approximately 300 μm to approximately 1000 μm, at a nozzle temperature of approximately 105°C to approximately 145°C, using a print speed of approximately 5 mm / s to approximately 30 mm / s.

[0300] Post-processing: The 3D-printed structures were immersed in distilled water for at least 16 hours to dissolve the polyethylene glycol from the printed material, resulting in a porous and flexible β-TCP / polycaprolactone composite. The scaffolds were then dried for at least 12 hours to ensure that residual moisture had evaporated from the porous scaffold before conjugation with the TBMP2 protein. The scaffolds were sterilized by immersion in a 70% ethanol solution for 2–4 hours and allowed to dry in a biosafety cabinet for approximately 12 hours. Images of example scaffolds are shown in Figures 13A–13D and 14A–14D. An example scaffold is the gyroid scaffold, which has an interconnected, isotropic porous structure without sharp corners or straight edges. These properties allow for sufficient fluid transport within the scaffold structure and are adaptable for cell attachment.

[0301] Ink formulations and scaffolds #17-#24 Inks #17 and #20-24 are printed onto scaffolds using a pellet-based method, as described for ink formulation and scaffold #16.

[0302] Ink Formulation and Scaffold #18 Method: To prepare a 16 cc batch of ink, 18 g of β-TCP powder, 6 g of caprolactone / glycolide copolymer (95:5) pellets, 3 g of polyethylene glycol (8,000 MW) flakes, and 3 g of polyethylene glycol (35,000 MW) flakes were added to a Teflon mixing vessel. The Teflon vessel was placed in a double asymmetric centrifugal mixer (FlackTek Speedmixer) and mixed at low intensity (300 rpm) for 2 minutes to homogenize the powder blend, followed by mixing at high rpm. The composition was mixed at high intensity (3500 rpm) for 1.5 minutes. During mixing, internal friction melted the caprolactone / glycolide copolymer and polyethylene glycol, transforming the ink into a viscous molten liquid. This liquid-phase mixing facilitated intimate dispersion of the β-TCP powder into the molten polymer blend. The molten ink was poured onto a glass plate to cool and smoothed with a spatula into a layer approximately 3 mm thick. The ink was allowed to cool for 10-15 minutes and then mixed at 3500 rpm for an additional 1.5 minutes. This mixing / cooling process was repeated a total of four times for 1.5 minutes at 3500 rpm. After the ink had cooled after the fourth and final mix, scissors were used to cut the ink into approximately 4 mm to 6 mm square pieces for pelletization.

[0303] Pellet production: An 850 μm diameter nozzle was attached to the metal barrel of a heated pneumatic extrusion device (e.g., an Allevi 3 bioprinter equipped with a 5 mL steel syringe barrel), and 3–4 cc of 4–6 mm square ink material was loaded into the barrel. The barrel was heated to a temperature of approximately 80°C to approximately 120°C, e.g., 100°C, and allowed to stand for 15 minutes. After 15 minutes, additional material was added to the barrel until it was filled, and pushed into the molten material with a spatula. The material was then allowed to stand for 10 minutes. After 10 minutes, the ink was confirmed to be completely melted. The air pressure of the pneumatic extrusion device was then set to approximately 50–100 psi, e.g., 55 psi. A platform was then provided below the extrusion tip as a substrate for extruding the pellets. In one example, the platform can be a smooth silicone sheet material. Using manually generated Marlin G-code, up to 120 pellets were extruded in a rectangular array, resulting in approximately equiaxed pellets with diameters of approximately 2 mm to approximately 3 mm.

[0304] 3D printing: The pellets were then loaded into the hopper of a Piocreat G5 FGF 3D printer. The material was printed using a nozzle diameter of approximately 300 μm to approximately 1000 μm, at a nozzle temperature of approximately 105°C to approximately 145°C, using a print speed of approximately 5 mm / s to approximately 30 mm / s.

[0305] Post-processing: The 3D-printed structures were soaked in distilled water for at least 16 hours to dissolve the polyethylene glycol from the printed material, creating a porous and flexible β-TCP and caprolactone / glycolide copolymer composite. The scaffolds were then dried for at least 12 hours to ensure that any residual moisture had evaporated from the porous scaffold before conjugation with the TBMP2 protein. The scaffolds were sterilized by immersion in a 70% ethanol solution for 2–4 hours and allowed to dry in a biosafety cabinet for approximately 12 hours. Images of example scaffolds are shown in Figures 15A–15B. After soaking and drying, the scaffolds contained 75% by weight β-TCP powder and 25% by weight caprolactone / glycolide copolymer (95:5).

[0306] Ink Formulation and Scaffold #19 Method: To prepare a 16 cc batch of ink, 18 g of β-TCP powder, 6 g of caprolactone / glycolide copolymer (90:10) flakes, 3 g of polyethylene glycol (8,000 MW) flakes, and 3 g of polyethylene glycol (35,000 MW) flakes were added to a Teflon mixing vessel. The Teflon vessel was placed in a double asymmetric centrifugal mixer (FlackTek Speedmixer) and mixed at low intensity (300 rpm) for 2 minutes to homogenize the powder blend, followed by mixing at high rpm. The composition was mixed at high intensity (3500 rpm) for 1.5 minutes. During mixing, internal friction melted the caprolactone / glycolide copolymer and polyethylene glycol, transforming the ink into a viscous molten liquid. This liquid-phase mixing facilitated intimate dispersion of the β-TCP powder into the molten polymer blend. The molten ink was poured onto a glass plate to cool and smoothed with a spatula into a layer approximately 3 mm thick. The ink was allowed to cool for 10-15 minutes and then mixed at 3500 rpm for an additional 1.5 minutes. This mixing / cooling process was repeated a total of four times for 1.5 minutes at 3500 rpm. After the ink had cooled after the fourth and final mix, scissors were used to cut the ink into approximately 4 mm to 6 mm square pieces for pelletization.

[0307] Pellet production: An 850 μm diameter nozzle was attached to the metal barrel of a heated pneumatic extrusion device (e.g., an Allevi 3 bioprinter equipped with a 5 mL steel syringe barrel), and 3–4 cc of 4–6 mm square ink material was loaded into the barrel. The barrel was heated to a temperature of approximately 80°C to approximately 120°C, e.g., 100°C, and allowed to stand for 15 minutes. After 15 minutes, additional material was added to the barrel until it was filled, and pushed into the molten material with a spatula. The material was then allowed to stand for 10 minutes. After 10 minutes, the ink was confirmed to be completely melted. The air pressure of the pneumatic extrusion device was then set to approximately 50–100 psi, e.g., 55 psi. A platform was then provided below the extrusion tip as a substrate for extruding the pellets. In one example, the platform can be a smooth silicone sheet material. Using manually generated Marlin G-code, up to 120 pellets were extruded in a rectangular array, resulting in approximately equiaxed pellets with diameters of approximately 2 mm to approximately 3 mm.

[0308] 3D printing: The pellets were then loaded into the hopper of a Piocreat G5 FGF 3D printer. The material was printed using a nozzle diameter of approximately 300 μm to approximately 1000 μm, at a nozzle temperature of approximately 105°C to approximately 145°C, using a print speed of approximately 5 mm / s to approximately 30 mm / s.

[0309] Post-processing: The 3D-printed structures were soaked in distilled water for at least 16 hours to dissolve the polyethylene glycol from the printed material, creating a porous and flexible β-TCP and caprolactone / glycolide copolymer composite. The scaffolds were then dried for at least 12 hours to ensure that any residual moisture had evaporated from the porous scaffold before conjugation with the TBMP2 protein. The scaffolds were sterilized by immersion in a 70% ethanol solution for 2–4 hours and allowed to dry in a biosafety cabinet for approximately 12 hours. Images of example scaffolds are shown in Figures 16A–16B. After soaking and drying, the scaffolds contained 75% by weight β-TCP powder and 25% by weight caprolactone / glycolide copolymer (90:10).

[0310] Structure Characteristics The physical properties of exemplary scaffolds made using inks #1-#12 and #18-#19 were measured and are summarized in Table 34.

[0311] The physical properties of exemplary scaffolds made using inks #16, #18, and #19 were measured and are summarized in Table 35.

[0312] [Table 34]

[0313] [Table 35]

[0314] The structures of this example are examined using Brunauer-Emmett-Teller (BET) surface area analysis by gas physisorption.

[0315] Compression tests are also conducted on the structure.

[0316] Example 3: Therapeutic Agents A chimeric polypeptide comprising a BMP therapeutic peptide linked to five β-tricalcium phosphate-binding peptides was expressed and purified using standard expression and purification methods. The chimeric polypeptide, designated tBMP-2, has the following sequence: MPIGSLLADTTHHRPWTVIGESTHHRPWSIIGESSHHKPFTGLGDTTHHRPWGILAESTHHKPWTASGAGGSEGGGSEGGTSGATGAGTSTSGGGASTGGGTGQAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR (SEQ ID NO: 434).

[0317] Example 4: Device fabrication The 3D printed construct of Example 2, "400 μm nozzle 3D printed flexible three-layer membrane," was combined with the tBMP-2 therapeutic agent of Example 3 to create a device. The scaffold was bound with tBMP-2 in a binding solution, and unbound tBMP-2 was washed off the scaffold. The resulting device contained a tBMP-2-bound scaffold. The device was lyophilized. Similar devices are prepared using the 3D printed constructs herein and growth factors.

[0318] Example 5: Animal Model One or more devices from Example 4 will be tested in animal models to demonstrate bone regeneration via μCT imaging and histological analysis. Indications include lumbar fusion (3D printed inserts for spinal fusion cages), posterolateral (PLF) spinal fusion (3D printed scaffolds spanning the transverse processes), segmental tibial defects (3D printed scaffolds based on patient CT data), and / or alveolar ridge augmentation (3D printed thin membranes).

[0319] The first study is a rabbit posterolateral fusion model. The purpose of this study is to evaluate the in vivo performance of the test device with varying growth factor concentrations and scaffold masses. Test groups will be evaluated for spinal fusion rate, new bone formation, and residual graft mass at 8 weeks post-implantation using plain radiography, micro-CT, biomechanical, and histological endpoints.

[0320] A 15 cm long skin incision was made in the dorsal midline of each rabbit, extending from L1 to the sacrum. The fascia and muscle were then dissected over the L5-L6 transverse processes (TP). The TP was then denuded using a high-speed burr machine. The test device was placed over the transverse processes, and the fascia and skin were closed and stapled. Pain medication was administered to ensure the animals' comfort, as approved by the IACUC. Animals were allowed free access to food and unrestricted movement within their cages. No postoperative immobilization devices were used. Over the following weeks, animals were closely monitored and given additional analgesics if signs of increased pain were observed in their movement, diet, disposition, or general activity. Rabbits were radiographed postoperatively and at 8 weeks. Micro-CT morphometric analysis was performed using regions of interest (ROIs) placed over the fixation sites to calculate bone area. The fixation sites of each animal were processed for histology at 8 weeks.

[0321] The second study is a sheep interbody fusion model to evaluate the test device in the interbody space of the sheep lumbar spine. Sheep undergo interbody fusion via a lateral approach. The implanted motion segment is stabilized with pedicle screws and rod fixation. After discectomy and endplate preparation, a PEEK interbody spacer is placed in the interbody space. The spacer is filled with the test device. Sheep are examined by in vivo MDCT at 4, 8, and 12 weeks postoperatively, followed by euthanasia at 6 months postoperatively. After euthanasia, μCT, manual palpation of the implanted motion segment, mineralization (plastic embedding), standard histology (H&E and trichrome), IHC (growth factors), and histomorphometry are performed.

[0322] Example 6: Cytotoxicity assay In vitro cytotoxicity studies were performed to measure cellular responses, particularly toxic effects, when exposed to extracts from the 3D printed scaffolds. The results of this in vitro study provide insight into the toxicity of the biomaterial components of the scaffolds (shown in Table 36) when implanted in the body. All cytotoxicity studies were performed using the guidelines of the International Organization for Standardization (ISO) 10993-5:2009 standard.

[0323] [Table 36]

[0324] Briefly, L929 mouse fibroblast cells were plated in 24-well plates at 1x10 5 Cells were seeded at a seeding density of 100 cells / well and placed in a humidified incubator at 37°C and 5% CO2 overnight. Sterile scaffold extracts (scaffolds shown in Table 36) were prepared by first soaking the scaffolds in culture medium for 24 hours and then adding these extracts to 24-well plates seeded with the L929 mouse fibroblast cell line. After 24 hours, visual inspection of the cells after exposure to the extract was used to determine whether there was a cytotoxic cellular response after exposing the cells to the scaffolds. Cells were considered cytotoxic if they detached, lysed, or showed a change in morphology. Microscopic images of cells exposed to the scaffolds in Table 36 are shown in Figure 17A. Cells were examined and a cytotoxicity score was calculated according to the following cytotoxicity scale defined in the ISO 10993-5:2009 standard: Scale 0 = non-cytotoxic, Scale 1 = weakly cytotoxic, Scale 2 = mildly cytotoxic, Scale 3 = moderately cytotoxic, and Scale 4 = severely cytotoxic.

[0325] High-density polyethylene (HDPE) (negative control) scored an average of less than 1, indicating non-cytotoxicity to mild cytotoxicity. 0.1% zinc diethyldithiocarbamate (ZDEC) (positive control) scored an average of 4, indicating severe cytotoxicity, with widespread cell detachment. 95:5 and 90:10 scored 0, indicating non-cytotoxicity. OT samples scored less than 1, indicating non-cytotoxicity to mild cytotoxicity. All 3D-printed scaffold samples showed minimal or no cell detachment, similar in appearance to the cell-only control samples. Image scores are shown in Figure 17B.

Claims

1. An ink formulation comprising approximately 55% to 65% by weight of β-tricalcium phosphate (βTCP), approximately 15% to 25% by weight of caprolactone / glycolido copolymer, approximately 5% to 15% of polyethylene glycol (PEG) with a molecular weight of approximately 500 g / mol to approximately 15,000 g / mol, and approximately 5% to 15% of PEG with a molecular weight of approximately 25,000 g / mol to approximately 50,000 g / mol.

2. The ink formulation according to claim 1, comprising approximately 60% by weight of βTCP, approximately 20% by weight of caprolactone / glycolido copolymer, approximately 10% by weight of PEG having a molecular weight of approximately 500 g / mol to approximately 15,000 g / mol, and approximately 10% of PEG having a molecular weight of approximately 25,000 g / mol to approximately 50,000 g / mol.

3. The ink formulation according to claim 2, comprising approximately 60% by weight of βTCP, approximately 20% by weight of caprolactone / glycolido copolymer, approximately 10% by weight of PEG having a molecular weight of approximately 8,000 g / mol, and approximately 10% of PEG having a molecular weight of approximately 35,000 g / mol.

4. The ink formulation according to claim 1, wherein the caprolactone / glycolido copolymer is caprolactone / glycolido copolymer (95:5).

5. The ink formulation according to claim 1, wherein the caprolactone / glycolido copolymer is caprolactone / glycolido copolymer (90:10).

6. A method for preparing a three-dimensional structure, comprising the step of performing additive manufacturing using the ink formulation described in claim 1.

7. The method according to claim 6, wherein the ink formulation is in the form of pellets.

8. The method according to claim 6, wherein the additive manufacturing includes thermal fusion granulation (FGF).

9. A structure prepared by additive manufacturing of the ink formulation described in claim 1.

10. A device comprising a therapeutic agent and the structure described in claim 9.

11. The device according to claim 10, wherein the therapeutic agent is non-covalently bonded to the structure.

12. The device according to claim 10, wherein the therapeutic agent contains a growth factor.

13. The device according to claim 12, wherein the growth factor is selected from Table 1.

14. The device according to claim 10, wherein the therapeutic agent comprises bone morphogenetic protein (BMP).

15. The device according to claim 10, wherein the therapeutic agent includes a targeted portion, and the targeted portion is non-covalently bonded to the structure.

16. The device according to claim 15, wherein the targeted portion comprises a polypeptide that is 80%, 85%, 90%, 95%, or 100% identical to any one of the sequences in Tables 2 to 3.

17. The device according to claim 10, wherein the therapeutic agent comprises a chimeric polypeptide having a sequence that is at least about 80%, 85%, 90%, 95%, or 100% identical to any one of sequence numbers 433 to 441.

18. A composition for use in a method for treating a bone defect in a subject requiring treatment of a bone defect, the method comprising the step of applying the structure described in claim 9 to the bone defect in the subject.

19. A composition for use in a method for treating a bone defect in a subject requiring treatment of a bone defect, the method comprising the step of applying the device according to any one of claims 10 to 17 to the bone defect in the subject.

20. A composition for use in the method of claim 18, wherein the bone defect is located in the spine.

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