Ceramic compositions and methods of use

Abstract

The present disclosure provides devices comprising a therapeutic agent bound to a printed three-dimensional structure. Also disclosed are ink formulations for three-dimensional printing. Additionally, provided herein are methods for manufacturing devices and their use, for example, in treating a condition in a subject in need thereof. The devices can be coated with a therapeutic agent, such as a therapeutic agent that promotes bone growth, and / or seeded with cells to generate devices for tissue replacement and transplantation.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Application No. 63 / 153,279, filed February 24, 2021, the entire contents of which are incorporated herein by reference.

[0003] Government rights

[0004] This invention was developed with government support under license number W81XWH-18-C-0182 granted by the U.S. Army Medical Material Command and license number W81XWH-20-C-0069 granted by the U.S. Army Medical Research Acquisition Activity. The government holds certain rights to this invention.

[0005] sequence list

[0006] This application contains a sequence list submitted via EFS-Web in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy was created on December 3, 2020, named 50222-709_601_SL.txt, and is 285,147 bytes in size. Background Technology

[0007] 3D printing is a manufacturing process that creates three-dimensional solid objects from digital files. In the additive process of 3D printing, objects are created by placing successive layers of material until the desired object is achieved, with a precision down to the micrometer. Combined with computer-aided design (CAD) software, 3D printing can produce complex functional shapes that are easily customized compared to traditional manufacturing methods.

[0008] Surgical implantation to promote tissue regeneration is a useful technique if the scaffold and / or other forms of graft material can match the mechanical properties of natural tissue. A variety of materials are available as inks for manufacturing porous 3D-printed structures for implantation, including materials that mimic tissue and enable it to regenerate. Inks with effective bioactivity and mechanical properties are required for natural tissue regeneration and, if used in 3D printing, can be customized and used for the repair of large tissue defects. Summary of the Invention

[0009] This disclosure provides ink formulations and methods for 3D printing scaffolds. Scaffolds that can be generated using such ink formulations and methods are also provided. Scaffolds can be coated with therapeutic agents (such as those promoting bone growth) and / or seeded with cells to generate devices for tissue replacement and transplantation. For some devices, the therapeutic agent can be tethered to the scaffold via targeted portions that interact with scaffold components (such as ceramic materials). Advantages of the materials and methods described herein include providing 3D-printed, customizable implants as well as more general-purpose objects such as strips, blocks, or cylinders. Because the implant is 3D printed, precise control over the implant geometry is possible. Therefore, implants printed with these inks are suitable for many different therapies, including long bone repair, spinal fusion, maxillofacial structures, etc. Different ink formulations result in materials with different properties in implantable devices, including varying porosity and flexibility.

[0010] The various inks and scaffolds disclosed herein are designed to enhance the accessibility of therapeutic agents and / or target portions to scaffold components (such as ceramic materials, like β-tricalcium phosphate). For example, the ink formulations described in Examples 1-2.

[0011] The various inks and methods disclosed herein are designed to improve the manufacturability of 3D-printed scaffolds by converting the inks into 1.75 mm diameter filaments. In filament form (rather than syringe-based bioprinting inks), larger quantities of material (e.g., 1 kg filaments wound on a spool) can be continuously prepared, stored, and used before the material needs to be refilled in the 3D printer. Additionally, fused filament fabrication (FFF) 3D printers typically have a larger usable build volume than syringe-based bioprinters and can print at faster nozzle speeds, both of which increase manufacturing throughput. Furthermore, a wide variety of FFF printers are available on the market compared to the relatively limited variety of syringe-based bioprinters. For example, ink formulations #5 and #6 were both formulated into 1.75 mm diameter filament materials and demonstrated in an FFF 3D printer, as further described in Examples 1-2.

[0012] The various inks and scaffolds disclosed herein are designed to optimize the bioabsorption properties of the scaffolds. For example, the copolymers used in ink formulation #2 (caprolactone / glycolic acid copolymer (95:5)), ink formulation #3 (caprolactone / glycolic acid copolymer (90:10)), and ink formulation #4 (poly(D,L-lactide-co-glycolic acid) copolymer (50:50)) have faster absorption rates than polycaprolactone. The absorption rates, from slowest to fastest, are as follows: polycaprolactone, polycaprolactone / polyglycolic acid copolymer (95:5), polycaprolactone / glycolic acid copolymer (90:10), poly(p-dioxanone / L-lactide copolymer (90:10), and poly(D,L-lactide-co-glycolic acid) copolymer (50:50).

[0013] In one aspect, this document provides a three-dimensional structure comprising about 50% to about 90% by weight of ceramic and about 10% to about 50% by weight of polymer, wherein the structure has a plurality of micropores. In some embodiments, the average pore size of the micropores is about 1 micrometer to about 500 micrometers, or about 50 micrometers to about 250 micrometers, or about 150 micrometers in diameter. In some embodiments, the micropores provide additional surface area to the structure for contact with therapeutic agents. In some embodiments, the micropores are formed after removing one or more pore-forming agents from the structure. In some embodiments, the pore-forming agent includes a second polymer, sugar, or salt, or a combination thereof. In some embodiments, the micropores are formed by releasing carbon dioxide from a foaming agent present during the fabrication of the structure. In some embodiments, the foaming agent includes sodium bicarbonate.

[0014] On the other hand, this paper provides a three-dimensional structure comprising about 50% to about 90% by weight of ceramic and about 10% to about 50% by weight of polymer, wherein the structure has an open porosity of about 15% to about 50%.

[0015] On the other hand, this article provides a three-dimensional structure comprising about 50% to about 90% by weight of ceramic and about 10% to about 50% by weight of polymer, wherein the density of the structure is about 1 g / cm3 to about 2 g / cm3.

[0016] On the other hand, this paper provides a three-dimensional structure comprising about 50% to about 90% by weight of ceramic and about 10% to about 50% by weight of polymer, wherein the pillar diameter of the structure is about 300 micrometers to about 600 micrometers.

[0017] In some embodiments of the structures provided herein, the ceramic comprises calcium phosphate, hydroxyapatite, fluorapatite, bone, silicate, or vanadate, or combinations thereof. In some embodiments of the structures provided herein, the polymer comprises polycaprolactone, caprolactone / glycolic acid copolymer, poly(D,L-lactide-co-glycolic acid) copolymer, or p-dioxanone / L-lactide copolymer, or combinations thereof. In some embodiments of the structures provided herein, the ceramic comprises calcium phosphate, hydroxyapatite, fluorapatite, bone, silicate, or vanadate, or combinations thereof, and the polymer comprises polycaprolactone, caprolactone / glycolic acid copolymer, poly(D,L-lactide-co-glycolic acid) copolymer, or p-dioxanone / L-lactide copolymer, or combinations thereof.

[0018] In some embodiments of the structures provided herein, the polymer comprises polycaprolactone, and the ceramic comprises calcium phosphate. In some embodiments, the structure comprises about 65% to about 85% calcium phosphate and about 15% to about 35% polycaprolactone by weight. In some embodiments, a structure is provided comprising about 65% to about 85% calcium phosphate and about 15% to about 35% polycaprolactone by weight.

[0019] In some embodiments of the structures provided herein, the polymer comprises a caprolactone / glycolic acid copolymer, and the ceramic comprises calcium phosphate. In some embodiments, the structure comprises about 65% to about 85% by weight of calcium phosphate and about 15% to about 35% by weight of caprolactone / glycolic acid copolymer. In some embodiments, a structure is provided comprising about 65% to about 85% by weight of calcium phosphate and about 15% to about 35% by weight of caprolactone / glycolic acid copolymer.

[0020] In some embodiments of the structures provided herein, the polymer comprises a poly(D,L-lactide-co-glycolic acid) copolymer or a p-dioxanone / L-lactide copolymer, and the ceramic comprises calcium phosphate. In some embodiments, the structure comprises about 65% to about 85% by weight of calcium phosphate and about 15% to about 35% by weight of poly(D,L-lactide-co-glycolic acid) copolymer or p-dioxanone / L-lactide copolymer. In some embodiments, a structure is provided comprising about 65% to about 85% by weight of calcium phosphate and about 15% to about 35% by weight of poly(D,L-lactide-co-glycolic acid) copolymer or p-dioxanone / L-lactide copolymer.

[0021] In some embodiments of the structure provided herein, the structure is formed from an ink comprising ceramics, polymers, and sacrificial pore-forming agents. In some embodiments of the structure provided herein, the structure is formed from filaments using an extrusion-based three-dimensional printing method.

[0022] A device is also provided, comprising the structures and therapeutic agents provided herein. In some embodiments, the therapeutic agent comprises bone morphogenetic protein (BMP). In some embodiments, the therapeutic agent comprises a targeting portion, and the targeting portion is non-covalently bound to a three-dimensional structure. In some embodiments, the targeting portion comprises a polypeptide having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with any of the sequences in Tables 2-3. In some embodiments, the therapeutic agent comprises a chimeric polypeptide comprising a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with any of SEQ ID NO:433-441.

[0023] In one aspect, this document provides an apparatus comprising: a therapeutic agent and a printed three-dimensional structure comprising about 50% to about 90% by weight of ceramic and about 10% to about 50% by weight of polymer. In some embodiments, the therapeutic agent is non-covalently bonded to the printed three-dimensional structure. In some embodiments, the ceramic comprises calcium phosphate, hydroxyapatite, fluorapatite, bone, silicate, or vanadate, or combinations thereof. In some embodiments, the ceramic comprises β-tricalcium phosphate (β-TCP). In some embodiments, the structure comprises about 70% to about 80% by weight of ceramic. In some embodiments, the structure comprises about 75% by weight of ceramic. In some embodiments, the polymer comprises polyester. In some embodiments, the polymer comprises polycaprolactone (PCL), polyglycolic acid, lactide, or poly(p-dioxanone) (PDS), or combinations thereof. In some embodiments, the polymer comprises polycaprolactone (PCL). In some embodiments, the polymer comprises polyglycolic acid. In some embodiments, the polymer comprises poly(p-dioxanone) (PDS). In some embodiments, the polymer comprises lactide. In some embodiments, the lactide comprises L-lactide. In some embodiments, the lactide comprises poly(D,L-lactide). In some embodiments, the polymer comprises a copolymer. In some embodiments, the copolymer comprises polycaprolactone (PCL) and polyglycolic acid. In some embodiments, the copolymer comprises about 90-95% PCL and about 5-10% polyglycolic acid. In some embodiments, the copolymer comprises a poly(D,L-lactide-co-glycolic acid) copolymer. In some embodiments, the copolymer comprises about 40-60% poly(D,L-lactide) and about 40-60% polyglycolic acid. In some embodiments, the copolymer comprises a PDS-glycolic acid copolymer. In some embodiments, the copolymer comprises about 90-95% PDS and about 5-10% glycolide. In some embodiments, the copolymer comprises a PDS-L-lactide copolymer. In some embodiments, the copolymer comprises about 90-95% PDS and about 5-10% L-lactide by molar weight. In some embodiments, the structure comprises about 15% to about 25% polymer by weight. In some embodiments, the structure comprises about 25% polymer by weight. In some embodiments, the printed three-dimensional structure is formed by an ink comprising about 30% to about 70% ceramic, about 10% to about 30% polymer by weight, and optionally one or more additional reagents. In some embodiments, the ink comprises about 50% to about 70% ceramic by weight. In some embodiments, the ink comprises about 60% ceramic by weight. In some embodiments, the ink comprises about 15% to about 25% ceramic by weight. In some embodiments, the ink comprises about 20% ceramic by weight.In some embodiments, the ink comprises one or more additional agents. In some embodiments, the ink comprises one or more additional agents at a weight of about 1% to about 30%. In some embodiments, one or more additional agents include additional polymers, particles, or foaming agents, or combinations thereof. In some embodiments, one or more additional agents include additional polymers. In some embodiments, the additional polymer includes polyethylene glycol. In some embodiments, the additional polymer is present in the ink at a weight of about 10% to about 30%. In some embodiments, one or more additional agents include particles. In some embodiments, the particles include sodium chloride, calcium chloride, sucrose, trehalose (e.g., α,α-trehalose dihydrate), or mannitol (e.g., D-mannitol), or combinations thereof. In some embodiments, the particles are present in the ink at a weight of about 1% to about 10%. In some embodiments, one or more additional agents include foaming agents. In some embodiments, the foaming agent includes baking powder (e.g., monocalcium phosphate, sodium bicarbonate, corn starch) and / or azodicarbonamide. In some embodiments, the particles are present in the ink at a weight of about 5% to about 20%. In some embodiments, the therapeutic agent comprises a mammalian growth factor or a functional portion thereof. In some embodiments, the therapeutic agent comprises one or more polypeptides selected from Table 1 or a functional portion thereof. In some embodiments, the therapeutic agent comprises bone morphogenetic protein (BMP). In some embodiments, the therapeutic agent comprises a targeting portion, and the targeting portion is non-covalently bound to the printed three-dimensional structure. In some embodiments, the targeting portion binds to the printed three-dimensional structure with an affinity of about 1 pM to about 100 μM. In some embodiments, the targeting portion comprises a polypeptide having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with any of the sequences in Tables 2-3. In some embodiments, the targeting portion comprises about 2, 3, 4, 5, 6, 7, 8, 9, or 10 sequences selected from the sequences in Tables 2-3. In some embodiments, the therapeutic agent includes a chimeric polypeptide comprising a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with any one of SEQ ID NO:433-441.

[0024] A method for treating a condition in a subject is also provided, the method comprising applying the device to the subject. In some embodiments, the condition includes bone defects, cartilage defects, soft tissue defects, tendon defects, fascia defects, ligament defects, organ defects, bone-tendon tissue defects, skin defects, osteochondral defects, osteoporosis, avascular necrosis, or congenital skeletal deformities, or combinations thereof. In some embodiments, the method includes spinal fusion. In some embodiments, spinal fusion includes posterior lumbar fusion (PLF) and / or intervertebral fusion. In some embodiments, the method includes bone repair, dental restoration, craniofacial restoration, ankle fusion, kyphosis correction, osteoplasty, scaphoid fracture repair, tendon-bone repair, rib reconstruction, subchondral bone repair, cartilage repair, or surgical implantation of a three-dimensional structure or device, or combinations thereof.

[0025] A formulation is also provided comprising about 30% to about 70% by weight of ceramic, about 10% to about 30% by weight of polymer, and optionally one or more additional agents. In some embodiments, the ceramic comprises calcium phosphate, hydroxyapatite, fluorapatite, bone, silicate, or vanadate, or combinations thereof. In some embodiments, the ceramic comprises β-tricalcium phosphate (β-TCP). In some embodiments, the ink comprises about 50% to about 70% by weight of ceramic. In some embodiments, the ink comprises about 60% by weight of ceramic. In some embodiments, the polymer comprises polyester. In some embodiments, the polymer comprises polycaprolactone (PCL), polyglycolic acid, lactide, or poly(p-dioxanone) (PDS), or combinations thereof. In some embodiments, the polymer comprises polycaprolactone (PCL). In some embodiments, the polymer comprises polyglycolic acid. In some embodiments, the polymer comprises poly(p-dioxanone) (PDS). In some embodiments, the polymer comprises lactide. In some embodiments, the lactide comprises L-lactide. In some embodiments, the lactide comprises poly(D,L-lactide). In some embodiments, the polymer comprises a copolymer. In some embodiments, the copolymer comprises polycaprolactone (PCL) and polyglycolic acid. In some embodiments, the copolymer comprises about 90-95% PCL and about 5-10% polyglycolic acid. In some embodiments, the copolymer comprises a poly(D,L-lactide-co-glycolic acid) copolymer. In some embodiments, the copolymer comprises about 40-60% poly(D,L-lactide) and about 40-60% polyglycolic acid. In some embodiments, the copolymer comprises a PDS-glycolic acid copolymer. In some embodiments, the copolymer comprises about 90-95% PDS and about 5-10% glycolide. In some embodiments, the copolymer comprises a PDS-L-lactide copolymer. In some embodiments, the copolymer comprises about 90-95% PDS and about 5-10% L-lactide by molar weight. In some embodiments, the ink comprises one or more additional agents. In some embodiments, the ink comprises about 1% to about 30% by weight of one or more additional agents. In some embodiments, one or more additional agents include additional polymers, particles, or foaming agents, or combinations thereof. In some embodiments, one or more additional agents include additional polymers. In some embodiments, the additional polymer includes polyethylene glycol. In some embodiments, the additional polymer is present in the ink at about 10% to about 30% by weight. In some embodiments, one or more additional agents include particles. In some embodiments, the particles include sodium chloride, calcium chloride, sucrose, trehalose (e.g., α,α-trehalose dihydrate), or mannitol (e.g., D-mannitol), or combinations thereof.In some embodiments, the particles are present in the ink at a weight of about 1% to about 10%. In some embodiments, one or more additional agents include a foaming agent. In some embodiments, the foaming agent includes baking powder (e.g., monocalcium phosphate, sodium bicarbonate, corn starch) and / or azodicarbonamide. In some embodiments, the particles are present in the ink at a weight of about 5% to about 20%.

[0026] A filament comprising the formulation is also provided. In some embodiments, the diameter of the filament is from about 1.5 mm to about 2 mm.

[0027] A method for preparing three-dimensional structures is also provided, which includes using the formulation in a three-dimensional printing method.

[0028] A three-dimensional structure prepared using an ink formulation is also provided. In some embodiments, the structure comprises about 50% to about 90% ceramic by weight. In some embodiments, the structure comprises about 50% to about 90% tricalcium phosphate by weight. In some embodiments, the structure comprises about 10% to about 50% polymer by weight. In some embodiments, the polymer comprises polyester. In some embodiments, the polymer comprises polycaprolactone (PCL), polyglycolic acid, lactide, or poly(p-dioxanone) (PDS), or combinations thereof. In some embodiments, the polymer comprises polycaprolactone (PCL). In some embodiments, the polymer comprises polyglycolic acid. In some embodiments, the polymer comprises poly(p-dioxanone) (PDS). In some embodiments, the polymer comprises lactide. In some embodiments, the lactide comprises L-lactide. In some embodiments, the lactide comprises poly(D,L-lactide). In some embodiments, the polymer comprises a copolymer. In some embodiments, the copolymer comprises polycaprolactone (PCL) and polyglycolic acid. In some embodiments, the copolymer comprises about 90-95% PCL and about 5-10% polyglycolic acid (PEG) by molar. In some embodiments, the copolymer comprises a poly(D,L-lactide-co-glycolic acid) copolymer. In some embodiments, the copolymer comprises about 40-60% poly(D,L-lactide) and about 40-60% PEG by molar. In some embodiments, the copolymer comprises a PDS-glycolic acid copolymer. In some embodiments, the copolymer comprises about 90-95% PDS and about 5-10% PEG by molar. In some embodiments, the copolymer comprises a PDS-L-lactide copolymer. In some embodiments, the copolymer comprises about 90-95% PDS and about 5-10% L-lactide by molar. In some embodiments, the structure comprises about 15% to about 25% polymer by weight. In some embodiments, the structure comprises about 25% polymer by weight.

[0029] A three-dimensional structure is also provided, comprising about 50% to about 90% ceramic by weight and about 10% to about 30% polymer by weight. In some embodiments, the ceramic comprises calcium phosphate, hydroxyapatite, fluorapatite, bone, silicate, or vanadate, or combinations thereof. In some embodiments, the ceramic comprises β-tricalcium phosphate (β-TCP). In some embodiments, the structure comprises about 65% to about 85% ceramic by weight. In some embodiments, the structure comprises about 70% to about 80% ceramic by weight. In some embodiments, the structure comprises about 75% ceramic by weight. In some embodiments, the polymer comprises polyester. In some embodiments, the polymer comprises polycaprolactone (PCL), polyglycolic acid, lactide, or poly(p-dioxanone) (PDS), or combinations thereof. In some embodiments, the polymer comprises polycaprolactone (PCL). In some embodiments, the polymer comprises polyglycolic acid. In some embodiments, the polymer comprises poly(p-dioxanone) (PDS). In some embodiments, the polymer comprises lactide. In some embodiments, lactide comprises L-lactide. In some embodiments, lactide comprises poly(D,L-lactide). In some embodiments, the polymer comprises a copolymer. In some embodiments, the copolymer comprises polycaprolactone (PCL) and polyglycolic acid. In some embodiments, the copolymer comprises about 90-95% PCL and about 5-10% polyglycolic acid. In some embodiments, the copolymer comprises a poly(D,L-lactide-co-glycolic acid) copolymer. In some embodiments, the copolymer comprises about 40-60% poly(D,L-lactide) and about 40-60% polyglycolic acid. In some embodiments, the copolymer comprises a PDS-glycolic acid copolymer. In some embodiments, the copolymer comprises about 90-95% PDS and about 5-10% glycolide. In some embodiments, the copolymer comprises a PDS-L-lactide copolymer. In some embodiments, the copolymer comprises about 90-95% PDS and about 5-10% L-lactide by molar weight. In some embodiments, the structure comprises about 15% to about 25% polymer by weight. In some embodiments, the structure comprises about 25% polymer by weight. In some embodiments, the structure is prepared by a three-dimensional printing method.

[0030] A method for manufacturing a three-dimensional structure is also provided, the method comprising depositing an ink formulation in a three-dimensional form. In some embodiments, the ink formulation includes the ink formulation described herein.

[0031] A method for treating the condition of a subject in need is also provided, which includes delivering a structure to the subject's organ or tissue.

[0032] A method for treating a condition in a subject is also provided, the method comprising delivering a structure manufactured by the method herein to an organ or tissue of the subject.

[0033] In some embodiments, the condition includes bone defects, cartilage defects, soft tissue defects, tendon defects, fascia defects, ligament defects, organ defects, bone-tendon tissue defects, skin defects, osteochondral defects, osteoporosis, ischemic necrosis, or congenital skeletal deformities, or combinations thereof. In some embodiments, the method includes spinal fusion. In some embodiments, spinal fusion includes posterior lumbar fusion (PLF) and / or intervertebral fusion. In some embodiments, the method includes bone repair, dental restoration, craniofacial restoration, ankle fusion, kyphosis correction, osteoplasty, scaphoid fracture repair, tendon-bone repair, rib reconstruction, subchondral bone repair, cartilage repair, or surgical implantation of a three-dimensional structure or device, or combinations thereof. In some embodiments, the method also includes treating the subject with a therapeutic agent.

[0034] A method for delivering a therapeutic agent to a subject in need is also provided, the method comprising delivering a device containing the therapeutic agent and a structure to an organ or tissue of the subject.

[0035] In some embodiments, the therapeutic agent comprises a mammalian growth factor or a functional portion thereof. In some embodiments, the therapeutic agent comprises one or more polypeptides selected from Table 1 or a functional portion thereof. In some embodiments, the therapeutic agent comprises bone morphogenetic protein (BMP). In some embodiments, the therapeutic agent comprises a targeting portion that is non-covalently bound to the structure. In some embodiments, the targeting portion binds to the printed three-dimensional structure with an affinity of about 1 pM to about 100 μm. In some embodiments, the targeting portion comprises a polypeptide having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with any of the sequences in Tables 5-6. In some embodiments, the targeting portion 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 includes a chimeric polypeptide or a portion thereof, the chimeric polypeptide comprising a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with any one of SEQ ID NO:433-441.

[0036] In some embodiments of the apparatus and / or structure herein, the density of the structure is from about 1 g / cm³ to about 2 g / cm³. In some embodiments of the apparatus and / or structure herein, the open porosity of the structure is from about 15% to about 50%. In some embodiments of the apparatus and / or structure herein, the structure according to any one of claims 91-139, wherein the diameter of the support pillars of the structure is from about 300 μm to about 600 μm.

[0037] Details of one or more embodiments of this disclosure are set forth in the accompanying drawings and the following description. Other features, objects, and advantages of this disclosure will become apparent from the description, the drawings, and the claims. Attached Figure Description

[0038] Figures 1A-1C The images are scanning electron microscope (SEM) images of exemplary 3D printed objects made using ink formulation #1 as described in Example 2. Figure 1A and Figure 1C This is an SEM image of the object's surface at an increased magnification. Figure 1B It is an SEM image of the side of the object.

[0039] Figures 2A-2C These are scanning electron microscope (SEM) images of exemplary 3D printed objects made using ink formulation #2 as described in Example 2. Figure 2A and Figure 2C This is an SEM image of the object's surface at an increased magnification. Figure 2B It is an SEM image of the side of the object.

[0040] Figures 3A-3C The images are scanning electron microscope (SEM) images of exemplary 3D printed objects made using ink formulation #3 as described in Example 2. Figure 3A and Figure 3C This is an SEM image of the object's surface at an increased magnification. Figure 3B It is an SEM image of the side of the object.

[0041] Figures 4A-4C These are scanning electron microscope (SEM) images of exemplary 3D printed objects made using ink formulation #4 as described in Example 2. Figure 4A and Figure 4C This is an SEM image of the object's surface at an increased magnification. Figure 4B It is an SEM image of the side of the object.

[0042] Figures 5A-5C The images are scanning electron microscope (SEM) images of exemplary 3D printed objects made using ink formulation #5 as described in Example 2. Figure 5A and Figure 5C This is an SEM image of the object's surface at an increased magnification. Figure 5B It is an SEM image of the side of the object.

[0043] Figures 6A-6C The images are scanning electron microscope (SEM) images of exemplary 3D printed objects made using ink formulation #6 as described in Example 2. Figure 6A and Figure 6CThis is an SEM image of the object's surface at an increased magnification. Figure 6B It is an SEM image of the side of the object.

[0044] Figures 7A-7C These are scanning electron microscope (SEM) images of exemplary 3D printed objects made using ink formulation #7 as described in Example 2. Figure 7A and Figure 7C This is an SEM image of the object's surface at an increased magnification. Figure 7B It is an SEM image of the side of the object.

[0045] Figures 8A-8C The images are scanning electron microscope (SEM) images of exemplary 3D printed objects made using ink formulation #8 as described in Example 2. Figure 8A and Figure 8C This is an SEM image of the object's surface at an increased magnification. Figure 8B It is an SEM image of the side of the object.

[0046] Figures 9A-9C The images are scanning electron microscope (SEM) images of exemplary 3D printed objects made using ink formulation #9 as described in Example 2. Figure 9A and Figure 9C This is an SEM image of the object's surface at an increased magnification. Figure 9B It is an SEM image of the side of the object.

[0047] Figures 10A-10C These are scanning electron microscope (SEM) images of exemplary 3D printed objects made using ink formulation #10 as described in Example 2. Figure 10A and Figure 10C This is an SEM image of the object's surface at an increased magnification. Figure 10B It is an SEM image of the side of the object.

[0048] Figures 11A-11C These are scanning electron microscope (SEM) images of exemplary 3D printed objects made using ink formulation #11 as described in Example 2. Figure 11A and Figure 11C This is an SEM image of the object's surface at an increased magnification. Figure 11B It is an SEM image of the side of the object.

[0049] Figures 12A-12C These are scanning electron microscope (SEM) images of exemplary 3D printed objects made using ink formulation #12 as described in Example 2. Figure 12A and Figure 12C This is an SEM image of the object's surface at an increased magnification. Figure 12B It is an SEM image of the side of the object. Detailed Implementation

[0050] This article provides various formulations and structures. These structures can be coated with tetherable proteins (e.g., growth factors) to achieve desired therapeutic effects, such as promoting bone growth after implantation of the tethered structure.

[0051] preparation

[0052] In one aspect, this document provides formulations for fabricating 3D-printed structures. As a non-limiting example, the formulation comprises ceramic materials such as calcium phosphate (e.g., tricalcium phosphate, β-tricalcium phosphate, α-tricalcium phosphate), hydroxyapatite, fluorapatite, bone (e.g., demineralized bone), glass (bioglass) (such as silicates, vanadates, and related ceramic minerals), or chelated divalent metal ions, or combinations thereof. In some embodiments, the ceramic material comprises β-tricalcium phosphate (β-TCP). In some embodiments, the formulation comprises approximately 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% of the formulation by weight. Ceramics of % 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%. For example, the formulation comprises about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70% ceramic by weight. 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), defoamers, dispersants, solvents, particles, foaming agents, and plasticizers.

[0053] In some embodiments, the formulation comprises one or more polymers, such as about 1, 2, 3, 4, or 5 polymers. Non-limiting examples of polymers include poly(ethylene oxide), poly(propylene oxide), polyethylene glycol (PEG), and polyesters. In some embodiments, the formulation comprises about 5-30% polymer by weight of the formulation. In some embodiments, the formulation comprises about 20-60% total polymer by weight. For example, the total polymer comprises two or more polymers in the formulation, wherein the total percentage of polymer in the formulation is about 20-60% by weight of the formulation. In some embodiments, the formulation comprises about 30% to about 50% total polymer by weight. As a non-limiting example, the formulation comprises about 35-45% total polymer by weight. In one example, the polymer includes poloxamer. Poloxamer is a block copolymer of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO). A non-limiting example of poloxamer is poloxamer 407, such as... F-127. In some cases, the formulation contains about 5-20%, 5-15%, 5-10%, 10-20%, 10-15%, or 15-20% poloxamer 407 by weight. As another example, the polymer includes polyethylene glycol (PEG). In some cases, the formulation contains 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% PEG by weight. For example, the formulation contains about 5-30%, or 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% PEG by weight. In some embodiments, a first PEG having a first molecular weight and a second PEG having a second molecular weight are present. In some embodiments, the molecular weight of the PEG can be from 500 g / mol to 25,000 g / mol.In some cases, the molecular weight of PEG is approximately 500 g / mol, approximately 1,000 g / mol, approximately 1,500 g / mol, approximately 20.00 g / mol, approximately 2,500 g / mol, approximately 3,000 g / mol, approximately 3,500 g / mol, approximately 4,000 g / mol, approximately 4,500 g / mol, approximately 5,000 g / mol, approximately 5,500 g / mol, approximately 6,000 g / mol, and approximately 6,500 g / mol. 00 g / mol, approximately 7,000 g / mol, approximately 7,500 g / mol, approximately 8,000 g / mol, approximately 8,500 g / mol, approximately 9,000 g / mol, approximately 9,500 g / mol, approximately 10,000 g / mol, approximately 10,500 g / mol, approximately 11,000 g / mol, approximately 11,500 g / mol, approximately 12,000 g / mol, approximately 12,500 g / mol, approximately 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, or about 25,000 g / mol. In a non-limiting exemplary embodiment, the formulation comprises PEG with a molecular weight of 1,500 g / mol. In another non-limiting exemplary embodiment, the formulation comprises PEG with a molecular weight of 8,000 g / mol. In another non-limiting exemplary embodiment, the formulation comprises PEG with a molecular weight of 20,000 g / mol. In a non-limiting exemplary embodiment, the formulation comprises about 15% by weight of a first PEG and about 15% by weight of a second PEG. As another example, the polymer includes poly(p-dioxanone) (PDS). In some cases, the formulation contains about 10-30%, 10-25%, 10-20%, 10-15%, 15-30%, 15-25%, 15-20%, 20-30%, 20-25%, or 25-30% PDS by weight. For example, the formulation contains about 15-25%, or 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% PDS by weight.As another example, the polymer includes polyesters. In some embodiments, the polyester includes biodegradable polyesters, such as polycaprolactone (PCL). In some cases, the formulation contains about 10-30%, 10-25%, 10-20%, 10-15%, 15-30%, 15-25%, 15-20%, 20-30%, 20-25%, or 25-30% PCL by weight. For example, the formulation contains about 15-25%, or 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% PCL by weight. In some cases, the formulation contains PCL with a molecular weight of 50,000 g / mol. In some embodiments, the polyester includes polyglycolic acid (PGA) or poly(glycolic acid). In some cases, the formulation contains approximately 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% of PGA. For example, the formulation contains about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% PGA by weight. In some cases, the molecular weight of the PGA is about 38,000-54,000. In some embodiments, the polyester comprises polylactide, such as poly(D,L-lactide).In some cases, the formulation contains approximately 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- Polylactide at concentrations of 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%. For example, the formulation contains about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15% polylactide by weight.

[0054] In some embodiments, the polymer includes a copolymer. In some cases, the copolymer comprises polyglycolic acid (PCL). In some cases, the copolymer comprises PCL and PCL. For example, the copolymer contains 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% (molar amounts). %, 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% of PCL and approximately 1 molar. -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% The copolymer comprises 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% polyglycolic acid (PLA). In some cases, the copolymer contains approximately 90-95% PCL and approximately 5-10% PDA on a molar basis. In some cases, the copolymer contains both PDS and PDA.For example, the copolymer contains approximately 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% (molar amounts). 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% of PDS and approximately 1 molar total. -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% The copolymer comprises 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% polyglycolic acid (PDS). In some cases, the copolymer contains about 90-95% PDS and about 5-10% PDS on a molar basis. In some cases, the copolymer comprises a PDS-glycolic acid copolymer. For example, the formulation contains about 10-30%, 10-25%, 10-20%, 10-15%, 15-30%, 15-25%, 15-20%, 20-30%, 20-25%, 25-30%, or 20% by weight of a PDS-glycolic acid copolymer. In some cases, the copolymer includes a poly(D,L-lactide-co-glycolic acid) copolymer. For example, the formulation contains about 10-30%, 10-25%, 10-20%, 10-15%, 15-30%, 15-25%, 15-20%, 20-30%, 20-25%, 25-30%, or 20% by weight of a poly(D,L-lactide-co-glycolic acid) copolymer. In some cases, the copolymer comprises lactide (e.g., poly(D,L-lactide)) and polyglycolic acid.For example, the copolymer contains 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% of lactide (e.g., poly(lactic acid)) on a molar basis. (D,L-lactide) 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% (on a molar basis) of glycolide. In some cases, the copolymer includes poly(D,L-lactide-co-glycolic acid) copolymers. For example, the formulation contains about 10-30%, 10-25%, 10-20%, 10-15%, 15-30%, 15-25%, 15-20%, 20-30%, 20-25%, 25-30%, or 20% by weight of a poly(D,L-lactide-co-glycolic acid) copolymer. In some cases, the copolymer comprises lactide (e.g., L-lactide) and PDS. For example, the copolymer contains 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% PDS and [other components] based on molar amounts. The copolymer comprises approximately 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%, and 60-65% lactide (e.g., L-lactide) on a molar basis. In some cases, the copolymer includes PDS-L-lactide copolymers. For example, the formulation contains about 10-30%, 10-25%, 10-20%, 10-15%, 15-30%, 15-25%, 15-20%, 20-30%, 20-25%, 25-30%, or 20% by weight of PDS-L-lactide copolymer. In some cases, the copolymer contains p-dioxanone.In some cases, the copolymer comprises p-dioxanone and lactide (e.g., L-lactide). For example, the copolymer contains approximately 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%, 8... (the text abruptly ends here, so the translation stops as well.) 5-95%, 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% of p-dioxanone and approximately 1-20 molar amounts. %, 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% lactide (e.g., L-lactide). In some cases, the copolymer contains about 85-95% p-dioxanone and about 5-15% lactide (e.g., L-lactide) on a molar basis.

[0055] In some cases, the copolymer is absorbed at a faster rate than the single polymer. For example, the copolymers used in exemplary embodiments of ink formulation #2 (polycaprolactone / polyglycolic acid copolymer (95:5)), ink formulation #3 (polycaprolactone / polyglycolic acid copolymer (90:10)), and ink formulation #4 (poly(D,L-lactide-co-glycolic acid) copolymer (50:50)) have a faster absorption rate than polycaprolactone. The absorption rates, from slowest to fastest, are as follows: polycaprolactone, polycaprolactone / polyglycolic acid copolymer (95:5), polycaprolactone / glycolic acid copolymer (90:10), poly(p-dioxanone / L-lactide copolymer (90:10), and poly(D,L-lactide-co-glycolic acid) copolymer (50:50).

[0056] In some embodiments, the formulation comprises two or more polymers. In some embodiments, 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% by weight 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% by weight of a second polymer. In non-limiting examples, the formulation comprises about 10-30% by weight of a first polymer and about 10-30% by weight of a 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% by weight of a 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% by weight of a second polymer. In some cases, the first and / or 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 a copolymer and the second polymer comprises PEG. The copolymer may comprise PCL and polyglycolic acid (e.g., 95 mol% polycaprolactone, 5 mol% polyglycolic acid; 90 mol% polycaprolactone, 10 mol% polyglycolic acid). The copolymer may comprise polylactide (e.g., poly(D,L-lactide)) and polyglycolic acid (e.g., 50 mol% poly(D,L-lactide), 50 mol% polyglycolic acid). The copolymer may comprise a PDS-glycolic acid copolymer (e.g., 90 mol% PDS, 10 mol% polyglycolic acid). The copolymer may comprise a PDS-L-lactide copolymer (e.g., 90 mol% PDS, 10 mol% L-lactide).

[0057] In some embodiments, the formulation comprises one or more particles. The particles may be pore-forming agents. The particles may be water-soluble. The particles may include salts and / or sugars. Non-limiting examples of particles include sodium chloride, calcium chloride, sucrose, trehalose (e.g., α,α-trehalose dihydrate), and mannitol (e.g., D-mannitol). In some cases, the particles 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% particles by weight. In some cases, the formulation comprises about 1-10%, or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% particles. In some embodiments, the average size of the particles or pore-forming agent is about 1 micrometer to about 500 micrometers in diameter. For example, approximately 1 micrometer to approximately 450 micrometers, approximately 1 micrometer to approximately 400 micrometers, approximately 1 micrometer to approximately 350 micrometers, approximately 1 micrometer to approximately 300 micrometers, approximately 1 micrometer to approximately 250 micrometers, approximately 1 micrometer to approximately 200 micrometers, approximately 1 micrometer to approximately 150 micrometers, approximately 50 micrometers to approximately 500 micrometers, approximately 50 micrometers to approximately 450 micrometers, approximately 50 micrometers to approximately 400 micrometers, approximately 50 micrometers to approximately 350 micrometers, approximately 50 micrometers to approximately 300 micrometers, approximately 50 micrometers to approximately 250 micrometers, approximately 50 micrometers to approximately 200 micrometers, approximately 50 micrometers to approximately 150 micrometers, approximately 100 micrometers to approximately 500 micrometers. 0 micrometers, about 100 micrometers to about 450 micrometers, about 100 micrometers to about 400 micrometers, about 100 micrometers to about 350 micrometers, about 100 micrometers to about 300 micrometers, about 100 micrometers to about 250 micrometers, about 100 micrometers to about 200 micrometers, about 100 micrometers to about 150 micrometers, about 150 micrometers to about 500 micrometers, about 150 micrometers to about 450 micrometers, about 150 micrometers to about 400 micrometers, about 150 micrometers to about 350 micrometers, about 150 micrometers to about 300 micrometers, about 150 micrometers to about 250 micrometers, or about 150 micrometers to about 200 micrometers in diameter. In some cases, the average size of the particles or pore-forming agent is about 50 micrometers to about 250 micrometers, about 60 micrometers to about 240 micrometers, about 70 micrometers to about 230 micrometers, about 80 micrometers to about 220 micrometers, or about 90 micrometers to about 210 micrometers in diameter.In some embodiments, the average size of the particles or pore-forming agent is from about 100 micrometers to about 200 micrometers, for example, from about 110 micrometers to about 190 micrometers, from about 120 micrometers to about 180 micrometers, from about 130 micrometers to about 170 micrometers, from about 140 micrometers to about 160 micrometers, or about 100 micrometers, about 110 micrometers, about 120 micrometers, about 130 micrometers, about 140 micrometers, about 150 micrometers, about 160 micrometers, about 170 micrometers, about 180 micrometers, about 190 micrometers, or about 200 micrometers in diameter. In some embodiments, the average size of the particles or pore-forming agent is about 150 micrometers in diameter. In some embodiments, after the particles or pore-forming agent are removed, the structure formed by the formulation has micropores that provide additional surface area for contact with the therapeutic agent compared to a structure formed with a formulation that does not contain particles or pore-forming agents. In some embodiments, the average pore size of the micropores in the structure is from about 1 micrometer to about 500 micrometers, or from about 50 micrometers to about 250 micrometers, or about 150 micrometers in diameter.

[0058] In some embodiments, the formulation comprises 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% by weight, or about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% of the foaming agent. In some cases, the foaming agent releases carbon dioxide groups during printing to create a foam structure that can increase the porosity of the 3D printed structure. Non-limiting examples of foaming agents include baking powders (e.g., monocalcium phosphate, sodium bicarbonate, corn starch) and azodicarbonamide. In some cases, the foaming agent includes sodium bicarbonate. In some cases, the formulation contains about 5-15%, or about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% sodium bicarbonate by weight. In some embodiments, the foaming agent provides micropores in the structure with an average diameter of about 1 micrometer to about 500 micrometers, or about 50 micrometers to about 250 micrometers, or about 150 micrometers.

[0059] In some embodiments, the formulation comprises a ceramic material (e.g., β-TCP) and a polymer. The polymer includes PEO, PPO, PDS, PEG, polyesters, copolymers, or combinations thereof. In some embodiments, the formulation comprises, by weight, 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%, 5 5-65%, 55-60%, 60-70%, 60-65%, or 65-70% of ceramic material (e.g., β-TCP), for example, about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70% of ceramic material (e.g., β-TCP) by weight. In some cases, the formulation contains about 5-20%, 5-15%, 5-10%, 10-20%, 10-15%, or 15-20% poloxamer 407 by weight. In some cases, the formulation contains about 10-30%, 10-25%, 10-20%, 10-15%, 15-30%, 15-25%, 15-20%, 20-30%, 20-25%, or 25-30% PEG by weight. In some cases, the formulation contains about 10-30%, 10-25%, 10-20%, 10-15%, 15-30%, 15-25%, 15-20%, 20-30%, 20-25%, or 25-30% PCL by weight. In some embodiments, the formulation contains about 10-30%, 10-25%, 10-20%, 10-15%, 15-30%, 15-25%, 15-20%, 20-30%, 20-25%, or 25-30% PDS by weight. In some embodiments, the formulation also contains an antifoaming agent. In some embodiments, the formulation also contains a dispersant. In some embodiments, the formulation also contains a solvent. In some embodiments, the formulation also contains a plasticizer. In some embodiments, the formulation also contains particles. In some cases, the formulation also contains a foaming agent.

[0060] In some embodiments, the formulation comprises a ceramic material (e.g., β-TCP) and particles. The particles may be water-soluble. Non-limiting examples of particles include salts and sugars, such as sodium chloride, calcium chloride, sucrose, trehalose (e.g., α,α-trehalose dihydrate), and mannitol (e.g., D-mannitol). In some embodiments, the formulation comprises, by weight, 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%, 5 5-65%, 55-60%, 60-70%, 60-65%, or 65-70% of ceramic material (e.g., β-TCP), for example, about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70% of ceramic material (e.g., β-TCP) by weight. 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% of particles by weight. In some embodiments, the formulation also comprises water. In some embodiments, the formulation also comprises a polymer. In some embodiments, the formulation also comprises an antifoaming agent. In some embodiments, the formulation also comprises a dispersant. In some embodiments, the formulation also comprises a solvent. In some embodiments, the formulation also comprises a plasticizer. In some cases, the formulation also comprises a foaming agent.

[0061] In some embodiments, the formulation comprises a ceramic material (e.g., β-TCP) and a blowing agent. Non-limiting examples of blowing agents include baking powders (e.g., monocalcium phosphate, sodium bicarbonate, corn starch) and azodicarbonamide. The blowing agent may include sodium bicarbonate. In some embodiments, the formulation comprises, by weight, 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%, 5 5-65%, 55-60%, 60-70%, 60-65%, or 65-70% of ceramic material (e.g., β-TCP), for example, about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70% of ceramic material (e.g., β-TCP) by weight. In some embodiments, the formulation 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% of a foaming agent by weight. In some embodiments, the formulation contains about 5-15%, or about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% sodium bicarbonate by weight. In some embodiments, the formulation also contains water. In some embodiments, the formulation also contains a polymer. In some embodiments, the formulation also contains an antifoaming agent. In some embodiments, the formulation also contains a dispersant. In some embodiments, the formulation also contains a solvent. In some embodiments, the formulation also contains a plasticizer. In some embodiments, the formulation also contains particles.

[0062] On the other hand, the formulation comprises a ceramic material and one or more polymers. In some embodiments, the formulation comprises about 30% to about 70% of a ceramic material (e.g., β-TCP). For example, 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%, 60-70%, 60-65%, 65-70%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70% ceramic materials (e.g., β-TCP). In some embodiments, the formulation comprises a first 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% by weight. The first polymer may be polycaprolactone (PCL). The first polymer may be poly(p-dioxanone) (PDS). 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% by weight. The second polymer may be polyethylene glycol (PEG). In a non-limiting embodiment, the formulation comprises about 30-70% by weight of ceramic, about 10-30% by weight of a first polymer, and about 10-30% by weight of a second polymer. For example, the formulation may comprise about 30-70% by weight of β-TCP, about 10-30% by weight of PCL, and about 10-30% by weight of PEG. As another example, the formulation may comprise about 30-70% by weight of β-TCP, about 10-30% by weight of PDS, and about 10-30% by weight of PEG.

[0063] In some further embodiments, the formulation comprises granules. The granules may be water-soluble. In some cases, the granules 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% granules by weight. For example, the formulation may contain about 30-70% β-TCP by weight, about 10-30% PCL by weight, about 10-30% PEG by weight, and about 1-10% particles by weight.

[0064] In some further embodiments, the formulation comprises a foaming agent. In some cases, the foaming 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% by weight, or about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% of a foaming agent. For example, the formulation may contain about 30-70% β-TCP by weight, about 10-30% PCL by weight, about 10-30% PEG by weight, and about 5-20% foaming agent by weight.

[0065] In some further embodiments, the polymer of the formulation is a copolymer, such as a copolymer of PCL and polyglycolic acid. For example, the copolymer comprises 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% (molar amounts). %, 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% of PCL and approximately 1 molar. -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% polyglycolic acid (PLA). In some cases, the copolymer contains about 90-95% PCL and about 5-10% PLA on a molar basis. In one example, the formulation may contain about 30-70% β-TCP by weight, about 10-30% PCL and a polyglycolic acid copolymer (e.g., 95 mol% polycaprolactone, 5 mol% polyglycolic acid) by weight, and about 10-30% PEG by weight. In another example, the formulation may contain about 30-70% β-TCP by weight, about 10-30% PCL and a polyglycolic acid copolymer (e.g., 90 mol% polycaprolactone, 10 mol% polyglycolic acid) by weight, and about 10-30% PEG by weight.

[0066] In some further embodiments, the polymer of the formulation is a copolymer, such as a PDS and a polyglycolic acid copolymer. For example, the copolymer comprises 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% (molar amounts). 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% of PDS and approximately 1 molar total. -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% polyglycolic acid (PDS). In some cases, the copolymer contains about 90-95% PDS and about 5-10% PDS on a molar basis. In one example, the formulation may contain about 30-70% β-TCP by weight, about 10-30% PDS and polyglycolic acid copolymer (e.g., 90 mol% PDS, 10 mol% polyglycolic acid) and about 10-30% PEG by weight.

[0067] In some further embodiments, the polymer of the formulation is a copolymer, such as a PDS and lactide copolymer. For example, the copolymer comprises 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% (molar amounts). 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% of PDS and approximately 1 molar total. -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% lactide. In some cases, the copolymer contains about 90-95% PDS and about 5-10% lactide on a molar basis. In one example, the formulation may contain about 30-70% β-TCP by weight, about 10-30% PDS and lactide copolymer by weight (e.g., 90 mol% PDS, 10 mol% lactide) and about 10-30% PEG by weight.

[0068] On the other hand, the formulation comprises a ceramic material and one or more polymers. In some embodiments, the formulation comprises about 30% to about 70% of a ceramic material (e.g., β-TCP). For example, 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%, 60-70%, 60-65%, 65-70%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70% ceramic materials (e.g., β-TCP). In some embodiments, the formulation comprises a first 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% by weight of the first polymer. The first polymer may be a copolymer. In some embodiments, the copolymer comprises lactide (e.g., poly(D,L-lactide)) and polyglycolic acid. In some embodiments, the copolymer comprises a poly(D,L-lactide-co-glycolic acid) copolymer. In some embodiments, the copolymer comprises about 50 mol% poly(D,L-lactide) and about 50 mol% polyglycolic acid. 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% by weight. The second polymer may be polyethylene glycol (PEG). In non-limiting embodiments, the formulation comprises about 30-70% by weight of ceramic, about 10-30% by weight of a first polymer, and about 10-30% by weight of a second polymer. For example, the formulation may comprise about 30-70% by weight of β-TCP, about 10-30% by weight of a copolymer, and about 10-30% by weight of PEG.

[0069] 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% of a ceramic material (e.g., β-TCP). For example, 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%, 60-70%, 60-65%, 65-70%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70% ceramic materials (e.g., β-TCP). In some embodiments, the formulation comprises a first 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% by weight of the first polymer. The first polymer may be a copolymer, such as a copolymer of dioxane and lactide (e.g., L-lactide).For example, the copolymer contains approximately 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%, and 85-96% (on a molar basis). p-Dioxane with concentrations of 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% and molar concentrations. 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%, 3-4%, 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% lactide. In some cases, the copolymer contains about 90-95% p-dioxanone and about 5-10% lactide by molar weight. 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% by weight. The second polymer may be polyethylene glycol (PEG). In non-limiting embodiments, the formulation comprises about 30-70% by weight of ceramic, about 10-30% by weight of a first polymer, and about 10-30% by weight of a second polymer. For example, the formulation may comprise about 30-70% by weight of β-TCP, about 10-30% by weight of a copolymer, and about 10-30% by weight of PEG. In one example, the formulation may contain about 30-70% β-TCP by weight, about 10-30% PDS and lactide copolymer (e.g., 90 mol% p-dioxanone, 10 mol% L-lactide) by weight, and about 10-30% PEG by weight.

[0070] On one hand, the formulation has low viscosity, which allows it to be extruded through a small-diameter nozzle during manufacturing. The nozzle diameter can be from about 240 μm to about 500 μm or from about 280 μm to about 450 μm, for example, about 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, or 500 μm. In a non-limiting exemplary embodiment, the formulation is melt-mixed in a dual asymmetric centrifugal mixer to produce a homogeneous liquid ink.

[0071] On one hand, the formulation has a high viscosity, which can be used during manufacturing to form the formulation into filaments. The filaments can then be used for filament fabrication.

[0072] In one aspect, the formulation is in filament form. For example, as further described in Example 2, the ink formulation is prepared into filaments for 3D printing structures on a fused filament fabrication (FFF) 3D printer. In some embodiments, the diameter of the filament formulation is 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, about 1.25 to about 1.5 mm. m, 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 diameter of the filament formulation is about 1.5 mm to about 2 mm, or about 1.5 mm, about 1.75 mm, or about 2 mm.

[0073] In one aspect, the formulation is in granular form. For example, as further described in Example 2, the ink formulation is prepared into granules for use in 3D structures. In some embodiments, the granules can be formed into filaments. In some embodiments, the granules can be formed into powder. In some embodiments, the length of the pellets is 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, 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 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 a non-limiting exemplary embodiment, the length of the pellets is 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 include various different shapes, including spears, rods, granules, blocks, particles, and particles of any suitable shape.

[0074] In one aspect, the formulation is in powder form. In some embodiments, the powder may be generated from granules. In a non-limiting exemplary embodiment, the components of the formulation are melt-mixed into a homogeneous ink and then cryogenically 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.

[0075] 3D printed structures

[0076] On the other hand, this article provides 3D-printed structures. These structures can be prepared using the formulations and / or manufacturing methods described herein.

[0077] In some embodiments, the three-dimensional structure has micropores. The micropores may be formed after the removal of particles or pore-forming agents. Micropores may also be formed by using a foaming agent during formulation. In some embodiments, the micropores provide additional surface area for contact with the therapeutic agent compared to structures lacking micropores. In a non-limiting example, the therapeutic agent includes a targeting portion that is non-covalently bonded to the ceramic material of the structure. In some embodiments, the average diameter of the micropores is from about 1 micrometer to about 500 micrometers. For example, approximately 1 micrometer to approximately 450 micrometers, approximately 1 micrometer to approximately 400 micrometers, approximately 1 micrometer to approximately 350 micrometers, approximately 1 micrometer to approximately 300 micrometers, approximately 1 micrometer to approximately 250 micrometers, approximately 1 micrometer to approximately 200 micrometers, approximately 1 micrometer to approximately 150 micrometers, approximately 50 micrometers to approximately 500 micrometers, approximately 50 micrometers to approximately 450 micrometers, approximately 50 micrometers to approximately 400 micrometers, approximately 50 micrometers to approximately 350 micrometers, approximately 50 micrometers to approximately 300 micrometers, approximately 50 micrometers to approximately 250 micrometers, approximately 50 micrometers to approximately 200 micrometers, approximately 50 micrometers to approximately 150 micrometers, approximately 100 micrometers to approximately 500 micrometers. 0 micrometer, about 100 micrometers to about 450 micrometers, about 100 micrometers to about 400 micrometers, about 100 micrometers to about 350 micrometers, about 100 micrometers to about 300 micrometers, about 100 micrometers to about 250 micrometers, about 100 micrometers to about 200 micrometers, about 100 micrometers to about 150 micrometers, about 150 micrometers to about 500 micrometers, about 150 micrometers to about 450 micrometers, about 150 micrometers to about 400 micrometers, about 150 micrometers to about 350 micrometers, about 150 micrometers to about 300 micrometers, about 150 micrometers to about 250 micrometers, or about 150 micrometers to about 200 micrometers in diameter. In some cases, the average diameter of the micropores is about 50 micrometers to about 250 micrometers, about 60 micrometers to about 240 micrometers, about 70 micrometers to about 230 micrometers, about 80 micrometers to about 220 micrometers, or about 90 micrometers to about 210 micrometers. In some embodiments, the average diameter of the micropores is from about 100 micrometers to about 200 micrometers, for example, from about 110 micrometers to about 190 micrometers, from about 120 micrometers to about 180 micrometers, from about 130 micrometers to about 170 micrometers, from about 140 micrometers to about 160 micrometers, or from about 100 micrometers, about 110 micrometers, about 120 micrometers, about 130 micrometers, about 140 micrometers, about 150 micrometers, about 160 micrometers, about 170 micrometers, about 180 micrometers, about 190 micrometers, or about 200 micrometers. In some embodiments, the average diameter of the micropores is about 150 micrometers.

[0078] In some embodiments, the density of the three-dimensional structure is approximately 1 g / cm³. 3 Approximately 3g / cm 3In some embodiments, the density of the three-dimensional structure is approximately 1 g / cm³. 3 Approximately 2g / cm 3 (For example, approximately 1, 1.1, 1.15, 1.2, 1.25, 1.3, 1.35, 1.4, 1.45, 1.5, 1.5, 1.55, 1.6, 1.65, 1.7, 1.75, 1.8, 1.85, 1.9, 1.95, or 2 g / cm³) 3 (or any value in between).

[0079] In some embodiments, the open porosity of the three-dimensional structure is 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%, for example, about 25%, 30%, 35%, or 40%, or any value between these values.

[0080] In some embodiments, the diameter of the struts of the three-dimensional structure is approximately 300 μm to 600 μm, approximately 325 μm to 600 μm, approximately 350 μm to 600 μm, approximately 375 μm to 600 μm, approximately 400 μm to 600 μm, approximately 425 μm to 600 μm, approximately 450 μm to 600 μm, approximately 475 μm to 600 μm, approximately 500 μm to 600 μm, approximately 525 μm to 600 μm, approximately 550 μm to 600 μm, approximately 300 μm to 575 μm, approximately 325 μm to 575 μm, approximately 350 μm to 575 μm, approximately 375 μm to 575 μm, and approximately 400 μm. Approximately 575 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 525 μm to approximately 575 μm, approximately 550 μm to approximately 575 μm, approximately 300 μm to approximately 550 μm, approximately 325 μm to approximately 550 μm, approximately 350 μm to approximately 550 μm, approximately 375 μm to approximately 550 μm, approximately 400 μm to approximately 550 μm, approximately 425 μm to approximately 550 μm, approximately 450 μm to approximately 550 μm, approximately 475 μm to approximately 550 μm, approximately 500 μm to approximately 550 μm, approximately 525 μm to approximately 550 μm, approximately 3 ...300 μm to approximately 550 μm, approximately 425 μm to approximately 550 μm, approximately 450 μm to approximately 550 μm, approximately 300 μm to approximately 550 μm, approximately 425 μm to approximately 550 μm, approximately 450 μm to approximately 550 μm, approximately 475 μm to approximately 550 μm, approximately 500 μm to approximately 550 μm, approximately 5 μm to about 525μm, about 325μm to about 525μm, about 350μm to about 525μm, about 375μm to about 525μm, about 400μm to about 525μm, about 425μm to about 525μm, about 450μm to about 525μm, about 475μm to about 525μm, about 500μm to about 525μm, about 300μm to about 500μm, about 325μm to about 500μm, about 350μm to about 500μm, about 375μm to about 500μm, about 400μm to about 500μm, about 425μm to about 500μm, about 450μm to about 500μm, about 475μm to about 500μm, about 3 00μm to about 475μm, about 325μm to about 475μm, about 350μm to about 475μm, about 375μm to about 475μm, about 400μm to about 475μm, about 425μm to about 475μm, about 450μm to about 475μm, about 300μm to about 450μm, about 325μm to about 450μm, about 350μm to about 450μm, about 375μm to about 450μm, about 400μm to about 450μm, about 425μm to about 450μm, about 300μm to about 400μm, about 325μm to about 400μm, about 350μm to about 400μm or about 375μm to about 400μm.

[0081] In some embodiments, the structure comprises a ceramic material, such as calcium phosphate. In some embodiments, the structure comprises approximately 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%, 60-10%... 0%, 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%, 63% Ceramic materials comprising 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some cases, the ceramic material is calcium phosphate, such as β-tricalcium phosphate (β-TCP).

[0082] In non-limiting examples, 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 poly(p-dioxanone) (PDS). In some cases, the structure has about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% polymer, such as PCL or PDS. Exemplary structures include those having the following components by weight: about 85-90% ceramic (e.g., β-TCP) and about 10-15% polymer (e.g., PCL or PDS); about 80-85% ceramic (e.g., β-TCP) and about 15-20% polymer (e.g., PCL or PDS); about 75-80% ceramic (e.g., β-TCP) and about 20-25% polymer (e.g., PCL or PDS); and about 70-75% ceramic (e.g., β-TCP) and about 25-30% polymer (e.g., PCL or PDS). The composition includes approximately 65-70% by weight of polymers (e.g., PCL or PDS) and approximately 30-35% by weight of ceramics (e.g., β-TCP); approximately 60-65% by weight of ceramics (e.g., β-TCP) and approximately 35-40% by weight of polymers (e.g., PCL or PDS); approximately 55-60% by weight of ceramics (e.g., β-TCP) and approximately 40-45% by weight of polymers (e.g., PCL or PDS); and approximately 50-55% by weight of ceramics (e.g., β-TCP) and approximately 45-50% by weight of... Polymers (e.g., PCL or PDS), approximately 90% by weight of ceramics (e.g., β-TCP) and approximately 10% by weight of polymers (e.g., PCL or PDS), approximately 89% by weight of ceramics (e.g., β-TCP) and approximately 11% by weight of polymers (e.g., PCL or PDS), approximately 88% by weight of ceramics (e.g., β-TCP) and approximately 12% by weight of polymers (e.g., PCL or PDS), approximately 87% by weight of ceramics (e.g., β-TCP) and approximately 13% by weight of polymers (e.g., PCL or PDS), and approximately 86% by weight of... The composition includes ceramics (e.g., β-TCP) and approximately 14% polymers (e.g., PCL or PDS); approximately 85% ceramics (e.g., β-TCP) and approximately 15% polymers (e.g., PCL or PDS) by weight; approximately 84% ceramics (e.g., β-TCP) and approximately 16% polymers (e.g., PCL or PDS) by weight; approximately 83% ceramics (e.g., β-TCP) and approximately 17% polymers (e.g., PCL or PDS) by weight; and approximately 82% ceramics (e.g., β-TCP) and approximately 18% polymers (e.g., PCL or PDS) by weight.PCL or PDS), approximately 81% ceramic (e.g., β-TCP) and approximately 19% polymer (e.g., PCL or PDS) by weight; approximately 80% ceramic (e.g., β-TCP) and approximately 20% polymer (e.g., PCL or PDS) by weight; approximately 79% ceramic (e.g., β-TCP) and approximately 21% polymer (e.g., PCL or PDS) by weight; approximately 78% ceramic (e.g., β-TCP) and approximately 22% polymer (e.g., PCL or PDS) by weight; approximately 77% ceramic (e.g., β-TCP) and approximately 23% polymer (e.g., PCL or PDS) by weight; approximately 76% ceramic (e.g., β-TCP) and approximately... 24% polymer (e.g., PCL or PDS), about 75% ceramic (e.g., β-TCP) and about 25% polymer (e.g., PCL or PDS) by weight, about 74% ceramic (e.g., β-TCP) and about 26% polymer (e.g., PCL or PDS) by weight, about 73% ceramic (e.g., β-TCP) and about 27% polymer (e.g., PCL or PDS) by weight, about 72% ceramic (e.g., β-TCP) and about 28% polymer (e.g., PCL or PDS) by weight, about 71% ceramic (e.g., β-TCP) and about 29% polymer (e.g., PCL or PDS) by weight, about 70% ceramic (e.g., β-TCP) by weight, and about 70% ceramic (e.g., PCL or PDS) by weight. For example, β-TCP and about 30% polymer (e.g., PCL or PDS), about 69% ceramic (e.g., β-TCP) and about 31% polymer (e.g., PCL or PDS) by weight, about 68% ceramic (e.g., β-TCP) and about 32% polymer (e.g., PCL or PDS) by weight, about 67% ceramic (e.g., β-TCP) and about 33% polymer (e.g., PCL or PDS) by weight, about 66% ceramic (e.g., β-TCP) and about 34% polymer (e.g., PCL or PDS) by weight, about 65% ceramic (e.g., β-TCP) and about 35% polymer (e.g., PCL or PDS) by weight, etc. The composition is approximately 64% ceramic (e.g., β-TCP) and approximately 36% polymer (e.g., PCL or PDS) by weight; approximately 63% ceramic (e.g., β-TCP) and approximately 37% polymer (e.g., PCL or PDS) by weight; approximately 62% ceramic (e.g., β-TCP) and approximately 38% polymer (e.g., PCL or PDS) by weight; approximately 61% ceramic (e.g., β-TCP) and approximately 39% polymer (e.g., PCL or PDS) by weight; approximately 60% ceramic (e.g., β-TCP) and approximately 40% polymer (e.g., PCL or PDS) by weight; and approximately 59% ceramic (e.g., β-TCP) and approximately 41% polymer (e.g., PCL or PDS) by weight.PCL or PDS), approximately 58% ceramic (e.g., β-TCP) and approximately 42% polymer (e.g., PCL or PDS) by weight; approximately 57% ceramic (e.g., β-TCP) and approximately 43% polymer (e.g., PCL or PDS) by weight; approximately 56% ceramic (e.g., β-TCP) and approximately 44% polymer (e.g., PCL or PDS) by weight; approximately 55% ceramic (e.g., β-TCP) and approximately 45% polymer (e.g., PCL or PDS) by weight; approximately 54% ceramic (e.g., β-TCP) by weight; The composition comprises approximately 46% by weight of TCP and about 46% by weight of polymers (e.g., PCL or PDS), approximately 53% by weight of ceramics (e.g., β-TCP) and about 47% by weight of polymers (e.g., PCL or PDS), approximately 52% by weight of ceramics (e.g., β-TCP) and about 48% by weight of polymers (e.g., PCL or PDS), approximately 51% by weight of ceramics (e.g., β-TCP) and about 49% by weight of polymers (e.g., PCL or PDS), and approximately 50% by weight of ceramics (e.g., β-TCP) and about 50% by weight of polymers (e.g., PCL or PDS).

[0083] This structure can be manufactured using 3D printing ink containing approximately 30-70% by weight β-TCP powder, approximately 10-30% by weight a first polymer, and approximately 10-30% by weight a second polymer. In some cases, the first polymer includes PCL. In some cases, the first polymer includes PDS. In some cases, the second polymer includes PEG. In some cases, the ink also contains approximately 1-10% by weight particulate matter (e.g., sucrose). In some cases, the ink also contains approximately 5-20% of a foaming agent (e.g., sodium bicarbonate).

[0084] In some embodiments, the density of the three-dimensional structure is approximately 1 g / cm³. 3 Approximately 2g / cm 3 or approximately 1.25 g / cm³ 3 Approximately 1.75 g / cm³ 3 In some embodiments, the open porosity of the three-dimensional structure is about 20% to about 40%, about 25% to about 35%, for example, about 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35%. In some embodiments, the strut diameter of the three-dimensional structure is about 400 μm to about 500 μm, about 400 μm to about 450 μm, or about 425 μm to about 450 μm.

[0085] In some embodiments, the density of the three-dimensional structure is approximately 1 g / cm³. 3 Approximately 2g / cm 3 or about 1g / cm 3Approximately 1.5 g / cm³ 3 In some embodiments, the open porosity of the three-dimensional structure is about 30% to about 50%, about 35% to about 45%, for example, about 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, or 45%. In some embodiments, the strut diameter of the three-dimensional structure is about 325 μm to about 425 μm, about 350 μm to about 400 μm, or about 360 μm to about 390 μm.

[0086] In some embodiments, the density of the three-dimensional structure is approximately 1 g / cm³. 3 Approximately 2g / cm 3 or about 1g / cm 3 Approximately 1.5 g / cm³ 3 In some embodiments, the open porosity of the three-dimensional structure is about 30% to about 50%, about 35% to about 45%, for example, about 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, or 45%. In some embodiments, the strut diameter of the three-dimensional structure is about 350 μm to about 450 μm, about 350 μm to about 400 μm, or about 380 μm to about 405 μm.

[0087] In non-limiting examples, 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% copolymer, such as polycaprolactone / polyglycolic acid copolymer (PCL / PGA, e.g., 90:10, 95:5), poly(D,L-lactide-co-glycolic acid) copolymer (PLGA, e.g., 50:50), PDS-glycolic acid copolymer (PDS / PGA, e.g., 90:10), PDS-L-lactide copolymer (PDS / PLA, e.g., 90:10), or p-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% of polymers such as PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide. Exemplary structures include those having the following components: approximately 85-90% ceramic (e.g., β-TCP) and approximately 10-15% polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide) by weight; approximately 80-85% ceramic (e.g., β-TCP) and approximately 15-20% polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide) by weight; approximately 75-80% ceramic (e.g., β-TCP) and approximately 20-25% polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide) by weight; approximately 70-75% ceramic (e.g., β-TCP) and approximately 25-30% polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide) by weight. L / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide), about 65-70% ceramic (e.g., β-TCP) and about 30-35% polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide) by weight, and about 60-65% ceramic (e.g., β-TCP) and about 35-4% polymer by weight. 0% polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide), about 55-60% ceramic (e.g., β-TCP) by weight, and about 40-45% polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide) by weight, and about 50-55% ceramic (e.g., β-TCP) by weight.The composition comprises approximately 45-50% by weight of β-TCP and about 45-50% by weight of polymers (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide), approximately 90% by weight of ceramics (e.g., β-TCP) and about 10% by weight of polymers (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide), approximately 89% by weight of ceramics (e.g., β-TCP) and about 11% by weight of polymers (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide), and approximately 88% by weight of ceramics (e.g., β-TCP). The composition comprises approximately 12% by weight of TCP and about 12% by weight of polymers (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide), approximately 87% by weight of ceramics (e.g., β-TCP) and about 13% by weight of polymers (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide), approximately 86% by weight of ceramics (e.g., β-TCP) and about 14% by weight of polymers (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide), and approximately 85% by weight of ceramics (e.g., β-TCP) and about 12% by weight of polymers (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide), and approximately 85% by weight of ceramics (e.g., β-TCP) and about 12% by weight of polymers (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide). 15% polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide), about 84% ceramic (e.g., β-TCP) and about 16% polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide) by weight, about 83% ceramic (e.g., β-TCP) and about 17% polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide) by weight, about 82% ceramic (e.g., β-TCP) and about 18% polymer (e.g., β-TCP) by weight. The composition includes approximately 81% by weight of ceramics (e.g., β-TCP) and approximately 19% by weight of polymers (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide), approximately 80% by weight of ceramics (e.g., β-TCP) and approximately 20% by weight of polymers (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide), and approximately 79% by weight of ceramics (e.g., β-TCP) and approximately 21% by weight of polymers (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide).PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide), approximately 78% by weight of ceramic (e.g., β-TCP) and approximately 22% by weight of polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide); approximately 77% by weight of ceramic (e.g., β-TCP) and approximately 23% by weight of polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide); approximately 76% by weight of ceramic (e.g., β-TCP) and approximately 24% by weight of polymer (e.g., PCL / PGA, PDS / PGA, PDS... The composition comprises approximately 75% by weight of ceramics (e.g., β-TCP) and approximately 25% by weight of polymers (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide), approximately 74% by weight of ceramics (e.g., β-TCP) and approximately 26% by weight of polymers (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide), and approximately 73% by weight of ceramics (e.g., β-TCP) and approximately 27% by weight of polymers (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide). The composition includes approximately 72% by weight of ceramics (e.g., β-TCP) and approximately 28% by weight of polymers (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide); approximately 71% by weight of ceramics (e.g., β-TCP) and approximately 29% by weight of polymers (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide); approximately 70% by weight of ceramics (e.g., β-TCP) and approximately 30% by weight of polymers (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide); and approximately 69% by weight of ceramics (e.g., ... The composition comprises approximately 31% by weight of ceramics (e.g., β-TCP) and about 31% by weight of polymers (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide), approximately 68% by weight of ceramics (e.g., β-TCP) and about 32% by weight of polymers (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide), approximately 67% by weight of ceramics (e.g., β-TCP) and about 33% by weight of polymers (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide), approximately 66% by weight of ceramics (e.g., β-TCP) and about 34% by weight of polymers (e.g., β-TCP).The composition comprises approximately 65% ​​by weight of ceramics (e.g., β-TCP) and approximately 35% by weight of polymers (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide); approximately 64% by weight of ceramics (e.g., β-TCP) and approximately 36% by weight of polymers (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide); and approximately 63% by weight of ceramics (e.g., β-TCP) and approximately 37% by weight of polymers (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide). The composition comprises approximately 62% by weight of ceramics (e.g., β-TCP) and approximately 38% by weight of polymers (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide), approximately 61% by weight of ceramics (e.g., β-TCP) and approximately 39% by weight of polymers (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide), and approximately 60% by weight of ceramics (e.g., β-TCP) and approximately 40% by weight of polymers (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide). The composition includes approximately 59% by weight of ceramics (e.g., β-TCP) and approximately 41% by weight of polymers (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide); approximately 58% by weight of ceramics (e.g., β-TCP) and approximately 42% by weight of polymers (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide); approximately 57% by weight of ceramics (e.g., β-TCP) and approximately 43% by weight of polymers (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide); and approximately 56% by weight of ceramics (e.g., β-TCP). The composition comprises approximately 44% by weight of ceramics (e.g., β-TCP) and about 44% by weight of polymers (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide), approximately 55% by weight of ceramics (e.g., β-TCP) and about 46% by weight of polymers (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide), approximately 53% by weight of ceramics (e.g., β-TCP) and about 47% by weight of polymers (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide), approximately 53% by weight of ceramics (e.g., β-TCP) and about 47% by weight of polymers (e.g., β-TCP).PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide; approximately 52% by weight of ceramic (e.g., β-TCP) and approximately 48% by weight of polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide); approximately 51% by weight of ceramic (e.g., β-TCP) and approximately 49% by weight of polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide); and approximately 50% by weight of ceramic (e.g., β-TCP) and approximately 50% by weight of polymer (e.g., PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide).

[0088] This structure can be manufactured using 3D printing ink containing approximately 30-70% by weight β-TCP powder, approximately 10-30% by weight a first polymer, and approximately 10-30% by weight a second polymer. In some cases, the first polymer includes PCL / PGA, PDS / PGA, PDS / PLA, PLGA, or p-dioxanone / L-lactide. In some cases, the second polymer includes PEG.

[0089] In some embodiments, the density of the three-dimensional structure is approximately 1 g / cm³. 3 Approximately 2g / cm 3 or approximately 1.25 g / cm³ 3 Approximately 1.75 g / cm³ 3 In some embodiments, the open porosity of the three-dimensional structure is about 15% to about 35%, about 20% to about 30%, for example, about 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%. In some embodiments, the strut diameter of the three-dimensional structure is about 350 μm to about 450 μm, about 375 μm to about 425 μm, or about 385 μm to about 415 μm.

[0090] In some embodiments, the density of the three-dimensional structure is approximately 1 g / cm³. 3 Approximately 2g / cm 3 or approximately 1.5g / cm 3 Approximately 2g / cm 3In some embodiments, the open porosity of the three-dimensional structure is about 15% to about 35%, about 20% to about 30%, for example, about 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%. In some embodiments, the strut diameter of the three-dimensional structure is about 500 μm to about 600 μm, about 550 μm to about 600 μm, or about 555 μm to about 585 μm.

[0091] In some embodiments, the density of the three-dimensional structure is approximately 1 g / cm³. 3 Approximately 2g / cm 3 or approximately 1.25 g / cm³ 3 Approximately 1.75 g / cm³ 3 In some embodiments, the open porosity of the three-dimensional structure is about 20% to about 40%, about 25% to about 35%, for example, about 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35%. In some embodiments, the strut diameter of the three-dimensional structure is about 400 μm to about 500 μm, about 450 μm to about 500 μm, or about 450 μm to about 475 μm.

[0092] In some embodiments, the components of the ink formulation described herein are modified to optimize the specific surface area. The surface area can be optimized for combination with a therapeutic agent. For example, the surface area of ​​the structure is approximately 0.2-2 m². 2 / g, to combine with BMP proteins (e.g., tBMP-2). In some embodiments, the surface area of ​​the structures described herein is about 0.2-2, 0.2-1.8, 0.2-1.6, 0.2-1.4, 0.2-1.2, 0.2-1, 0.2-0.8, 0.2-0.6, 0.2-0.4, 0.4-2, 0.4-1.8, 0.4-1.6, 0.4-1.4, 0.4-1.2, 0.4-1, 0.4-0.8, 0.4-0.6, 0.6-2, 0.6-1.8, 0.6-1.6, 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 1.8-2m 2 / g. In some embodiments, the surface area is calculated by Brunauer-Emmett-Teller (BET) via gas physical adsorption.

[0093] In some embodiments, the components of the ink formulation described herein are varied to optimize the absorption rate of one or more materials of the scaffold. For example, polymers are selected based on absorption rate. The absorption rates, from slowest to fastest, are as follows: polycaprolactone, polycaprolactone / polyglycolic acid copolymer (95:5), polycaprolactone / glycolic acid copolymer (90:10), polydioxanone / L-lactide copolymer (90:10), and poly(D,L-lactide-co-glycolic acid) copolymer (50:50).

[0094] Manufacturing method

[0095] On the other hand, it provides a method for manufacturing structures using 3D printing technology.

[0096] In some embodiments, the method includes syringe-based melt extrusion bioprinting. Exemplary inks used in this method may be of low viscosity to allow for ink extrusion through a narrow nozzle. Non-limiting exemplary methods for manufacturing using this method are described in Example 2, for example, regarding printing ink formulations #1, #2, #3, and #4.

[0097] On one hand, the method is based on extrusion, which includes a 3D printing method that extrudes material from a nozzle.

[0098] In some embodiments, extrusion-based methods include bioprinting (injector-based pneumatic printing) or fused particle manufacturing (FGF), in which a pellet of raw material is fed from a hopper into a microscrew extruder that melts and ejects the material through a fine nozzle. In some embodiments, the ink forms small (e.g., 2-5 mm) pellets or granules.

[0099] In some embodiments, the method includes fused filament fabrication (FFF). Exemplary inks used in this method can be formed into filaments for printing on an FFF 3D printer. Non-limiting exemplary methods for fabrication using this method are described in Example 2, for example, regarding printing ink formulations #5 and #6.

[0100] In some implementations, the method includes granulation fused deposition modeling. Fused deposition modeling (FDM) is an additive manufacturing process. Three-dimensional objects are formed by extruding and depositing individual layers of thermoplastic material. FDM involves molten extrusion of filament material through a heated nozzle and depositing it as a thin solid layer on a platform. Thermoplastic polymer material is fed into a temperature-controlled FDM extruder head and heated to a semi-liquid state. The FDM extruder head then precisely extrudes and deposits an ultrathin layer of material onto a substrate. The material is cured and laminated onto the previous layer. In this way, parts are manufactured layer by layer, where each layer is constructed by extruding small material beads (called roads) in a specific pattern such that the layer is covered by adjacent roads. After each layer is completed, the extruder head height is increased and subsequent layers are built to construct the part. Typically, FDM is used to manufacture solid models. To create porous structures, grating-filled gaps have positive values, which are used to impart channels within the build-up layers. The channels are arranged in a regular pattern and interconnected even in three-dimensional space. Layer-by-layer manufacturing allows for the design of varying pore morphologies on the scaffold structure.

[0101] In some embodiments, the method includes selective laser sintering (SLS). Selective laser sintering (SLS) is a process in which a distributor deposits layers of powdered material onto a target region. A laser control mechanism is present, typically including a computer storing an article design. The laser control mechanism modulates and moves a laser beam to selectively irradiate the powder layer within defined boundaries of the design, melting the powder that the laser beam falls upon. This is done to selectively sinter continuous powder layers. The method produces a complete article consisting of multiple layers sintered together.

[0102] In some embodiments, after 3D printing, the resulting object is immersed in water to dissolve certain components of the ink, such as PEG, particles (e.g., pore-forming agents, sucrose), foaming agents (e.g., sodium bicarbonate), or combinations thereof. The structure can then be dried, sterilized, treated with therapeutic agents as described elsewhere herein, or combinations thereof.

[0103] Any of the 3D-printed structures described herein may be coated with a tetherable protein (e.g., tBMP2). After completing the structure using any of the methods discussed herein, the structure may be washed in acidic sodium acetate buffer. Washing may be performed once, twice, or multiple times. After washing, the structure may be incubated for two hours in sodium acetate buffer containing 1 mg / mL of tBMP2 protein. Tetherable tBMP2 binds as a monolayer to the β-TCP surface of the implantable structure.

[0104] In other embodiments, the ink formulations discussed herein may include a photosensitive resin mixed with ceramic powder for digital light processing (DLP), an additive manufacturing technique that is faster than robotic casting 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 ≤10 μm particle size), one or more crosslinked acrylates or methacrylates (e.g., polyethylene glycol diacrylate, polycaprolactone methacrylate), plasticizers to reduce resin viscosity (e.g., water), and dispersants to promote the breakdown of powder agglomerates (e.g., [missing information]). The components include a photoinitiator (e.g., lithium phenyl-2,4,6-trimethylbenzoylphosphine) that initiates the photocrosslinking reaction, and a light absorber (e.g., tartrazine) that maintains high xy resolution. Once the resin formulation is prepared by asymmetric centrifugal mixing of the components, the ink is exposed layer by layer to the DLP image, allowing the luminescent pixels to selectively cure upon exposure to light. Once the implantable structure has been constructed layer by layer, it can be heat-treated to burn off the contained polymer and densify the ceramic (e.g., a resin containing polyethylene glycol diacrylate), or left as is, to obtain a flexible ceramic / polymer composite implant (e.g., a resin containing polycaprolactone methacrylate).

[0105] Device

[0106] On the other hand, devices and kits are provided that include the 3D-printed structures and therapeutic agents described herein. In some embodiments, the device includes a therapeutic agent attached to, dispersed within, or otherwise combined with the 3D-printed structure. As used herein, the therapeutic agent includes multiple therapeutic agents, such as two, three, four, or five therapeutic agents.

[0107] Therapeutic agents

[0108] In some embodiments, the therapeutic agent includes a mammalian growth factor or a functional portion thereof. The mammalian growth factor can be a osteoinductive molecule capable of initiating and enhancing the bone repair process. The functional portion of the mammalian growth factor is the region with the 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 is capable of initiating and / or enhancing bone repair. The functional portion of the mammalian growth factor may have osteogenic activity.

[0109] This article describes non-limiting examples of mammalian growth factors. In some cases,Mammalian growth factors include: epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-1), fibroblast growth factor (FGF), fibroblast growth factor 2 (FGF2), fibroblast growth factor 18 (FGF18), transforming growth factor α (TGF-α), transforming growth factor β (TGF-β), transforming growth factor β1 (TGF-β1), transforming growth factor β3 (TGF-β3), osteoblast protein 1 (OP-1), osteoblast protein 2 (OP-2), osteoblast protein 3 (OP-3), bone morphogenetic protein 2 (BMP-2), bone morphogenetic protein 3 (BMP-3), and bone morphogenetic protein 4 (BMP-4). BMP-4, BMP-5, BMP-6, BMP-7, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-15, Dentin phosphoprotein (DPP), Plant-associated growth factor (Vgr), Growth differentiation factor 1 (GDF-1), Growth differentiation factor 3 (GDF-3), Growth differentiation factor 5 (GDF-5), Growth differentiation factor 6 (GDF-6), and so on. 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)-associated apoptosis-inducing ligand (TRAIL), WNT family member 1 (WNT1), WNT family member 2 (WNT2), WNT family member 2B (WNT2B), WNT family member 3 (WNT3), WNT family members 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 its mature peptides or functional portions.

[0110] In some embodiments, the mammalian growth factor is human growth factor. Table 1 provides non-limiting examples of human growth factors and their mature peptides and / or functional portions. In some embodiments, the mammalian growth factor comprises a sequence having at least 70% identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity) with any of the sequences in Table 1 and has osteogenic activity. In some embodiments, amino acids conserved across different species in the mammalian growth factor may be important for osteogenic activity and may not be mutated, while non-conserved amino acids across different species in the mammalian growth factor may not be important for osteogenic activity and may be mutated.

[0111] In some embodiments, the mammalian growth factor includes BMP-2. In some embodiments, the mammalian growth factor is a mature peptide of BMP-2 (e.g., without a signal sequence). In some embodiments, the mammalian growth factor includes a functional portion of BMP-2. In some embodiments, the functional portion of BMP-2 includes a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with the following sequence: QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR (SEQ ID NO: 454). In some embodiments, the mammalian growth factor includes a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO: 454. In some embodiments, the mammalian growth factor comprises a sequence having at least about 90% identity with SEQ ID NO:454. In some embodiments, the mammalian growth factor includes SEQ ID NO:454.

[0112] In some embodiments, the mammalian growth factor is a non-human mammalian growth factor. The non-human mammalian growth factor may be homologous to human growth factors, such as one or more of the human growth factors listed in Table 1. In some embodiments, if, as determined using the NCBI Blast alignment algorithm as of the date of this application, the non-human mammalian growth factor is homologous to human growth factors if it has at least about 80% identity with human mammalian growth factors. In some cases, the coverage is at least about 90%. In some embodiments, if, as determined using the NCBI Blast alignment algorithm as of the date of this application, the non-human mammalian growth factor is at least about 80% positive compared to human mammalian growth factors, then the non-human mammalian growth factor is homologous to human growth factors. In some cases, the coverage is at least about 90%. In some implementations, if the E value of a non-human mammalian growth factor compared with human growth factor using NCBI Blast as of the date of this application is less than about 1E-40, at least about 1E-50, 1E-60, 1E-70, or 1E-10, and the query coverage is at least about 90%, then the non-human mammalian growth factor is homologous to human growth factor.

[0113] Table 1. Therapeutic growth factors

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131] Targeted portion

[0132] In some embodiments, the device or kit includes a targeting portion that tethers a therapeutic agent to the structure. In some embodiments, the targeting portion is connected to the therapeutic agent, and the portion is non-covalently bound to the structure. As a non-limiting example, the targeting portion is covalently connected to the therapeutic agent via peptide bonds. For example, the targeting portion comprises a targeting peptide that is linked to the therapeutic agent via peptide bonds.

[0133] In some embodiments, the targeting portion has an affinity for the structure or a component of the structure, for example, an affinity for the ceramic material of the structure (such as calcium phosphate). In some embodiments, the dissociation constant (KD) of the binding between the targeting portion and the structure or its components 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 portion can 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.

[0134] In some embodiments, the targeting portion includes one or more targeting peptides, each binding to the structure. In some embodiments, the targeting peptide binds to a ceramic material of the structure. For example, the targeting peptide binds to calcium phosphate (e.g., tricalcium phosphate, β-tricalcium phosphate, α-tricalcium phosphate), hydroxyapatite, fluorapatite, bone (e.g., demineralized bone), glass (bioglass) (such as silicates, vanadates, and related ceramic minerals), or chelated divalent metal ions, or combinations thereof. In some embodiments, the targeting peptide includes two or more targeting peptides. In some embodiments, the two or more targeting peptides do not exceed about 50, 45, 40, 35, 30, 25, 20, 15, or 10 targeting peptides. In some embodiments, the two or more targeting peptides are 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.

[0135] In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:1. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:2. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:3. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:4. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:5. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:6. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:7. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:8. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:9. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:10. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:11. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:12. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:13. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:14. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:15.In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:16. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:17. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:18. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:19. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:20. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:21. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:22. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:23. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:24. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:25. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:26. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:27. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:28. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:29.In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:30. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:31. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:32. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:33. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:34. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:35. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:36. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:37. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:38. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:39. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:40. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:41. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:42. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:43.In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:44. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:45. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:46. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:47. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:48. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:49. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:50. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:51. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:52. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:53. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:54. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:55. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:56. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:57.In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:58. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:59. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:60. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:61.

[0136] Table 2. Targeted Peptides

[0137]

[0138]

[0139]

[0140]

[0141] In some embodiments, the targeting peptide comprises one or more sequences from Table 2. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with the sequences in Table 2.

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148] In some embodiments, the targeting peptide comprises one or more sequences from Table 3. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with the sequences in Table 3.

[0149] Additional targeting peptides that can be used in this disclosure include any one of SEQ ID NO:1 to SEQ ID NO:558 of US 7,572,766. In some embodiments, the targeting peptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with any one of SEQ ID NO:1 to SEQ ID NO:558 of US 7,572,766.

[0150] In some embodiments, the device or kit includes a chimeric polypeptide comprising a targeting peptide and a targeting moiety. In some cases, the chimeric polypeptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:433 (ASGAGGSEGGGSEGGTSGATGAGTSTSGGGASTGGGTGQAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR). In some cases, the chimeric polypeptide contains a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:434 (MPIGSLLADTTHHRPWTVIGESTHHRPWSIIGESSHHKPFTGLGDTTHHRPWGILAESTHHKPWTASGAGGSEGGGSEGGTSGATGAGTSTSGGGASTGGGTGQAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR). In some cases, the chimeric polypeptide contains a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:435 (LLADTTHHRPWTVIGESTHHRPWSIIGESSHHKPFTGLGDTTHHRPWGILAESTHHKPWTASGAGGSEGGGSEGGTSGATGAGTSTSGGGASTGGGTGQAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR).In some cases, the chimeric polypeptide contains a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:436 (VIGESTHHRPWSIIGESSHHKPFTGLGDTTHHRPWGILAESTHHKPWTASGAGGSEGGGSEGGTSGATGAGTSTSGGGASTGGGTGQAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR). In some cases, the chimeric polypeptide contains a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:437 (IIGESSHHKPFTGLGDTTHHRPWGILAESTHHKPWTASGAGGSEGGGSEGGTSGATGAGTSTSGGGASTGGGTGQAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR). In some cases, the chimeric polypeptide contains a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:438 (GLGDTTHHRPWGILAESTHHKPWTASGAGGSEGGGSEGGTSGATGAGTSTSGGGASTGGGTGQAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR).In some cases, the chimeric polypeptide contains a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:439 (ILAESTHHKPWTASGAGGSEGGGSEGGTSGATGAGTSTSGGGASTGGGTGQAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR). In some cases, the chimeric polypeptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:440 ((X)QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAF YCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR), wherein X comprises a targeting peptide and optionally comprises a linker. For example, the targeting peptide comprises one or more of SEQ ID NO:1-41. In some cases, the chimeric polypeptide comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:441 ((X)ASGAGGSEGGGSEGGTSGATGAGTSTSGGGASTGGGTGQAKH KQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR), wherein X comprises a targeting peptide and optionally comprises a linker. For example, the targeting peptide comprises one or more of SEQ ID NO:1-41.

[0151] In some implementations, the therapeutic agent is not attached to the structure using a targeting portion. For example, the therapeutic agent may interact with the structure via non-covalent bonds. The therapeutic agent may be attached to the structure via hydrogen bonds, ionic bonds, hydrophobic interactions, or van der Waals forces. The therapeutic agent may also be attached to the structure using covalent bonds. Examples of methods using covalent attachment include chemical linkers and spacer regions for modifying active groups (e.g., amines, thiols, and carbohydrates) within proteins.

[0152] In some embodiments, a device is provided that includes a structure seeded with cells. Non-limiting examples of cells include osteoblasts and other bone cells, chondrocytes, and meniscus cells. In some cases, cells can be added to an intact implantable structure.

[0153] Equipment manufacturing

[0154] This document also provides methods for manufacturing an apparatus comprising a structure and a therapeutic agent. Some methods include: (a) providing a first solution of the therapeutic agent (e.g., a chimeric peptide comprising the therapeutic agent and a targeting portion), (b) providing a 3D structure, and (c) combining (a) and (b). In some embodiments, the therapeutic agent is present in the first solution at a concentration of about 0.25-1.5, 0.25-1.25, 0.25-1, 0.25-0.75, 0.25-0.5, 0.5-1.5, 0.5-1.25, 0.5-1, 0.5-0.75, 0.75-1.5, 0.75-1.25, 0.75-1, 1-1.5, 1-1.25, 1.25-1.5, 0.25, 0.5, 1, 1.25, or 1.5 mg / mL. In some methods, step (c) includes incubating the first solution and the structure at approximately 10-240, 10-180, 10-120, 20-240, 20-180, 20-120, 30-240, 30-180, 30-120, 40-240, 40-180, 40-120, 50-240, 50-180, 50-120, 60-240, 60-180, 60-120, 70-240, 70-180, 70-120, 80- 240, 80-180, 80-120, 90-240, 90-180, 90-120, 100-240, 100-180, 100-120, 110-240, 110-180, 110-120, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, or 240 minutes. In some methods, step (c) includes incubating the first solution and structure under movement such as rotation and / or shaking (e.g., using a plate shaker). In some cases, the first solution contains a buffer solution. For example, the buffer solution contains sodium acetate and acetic acid. In some cases, the pH of the first solution is about 4 to about 5, or about 4, 4.1, 4.15, 4.2, 4.25, 4.3, 4.35, 4.4, 4.45, 4.5, 4.45, 4.6, 4.65, 4.7, 4.75, 4.8, 4.85, 4.9, 4.95, or 5. In some cases, the first solution contains salt.For example, the first solution contains sodium chloride, such as approximately 50-150, 50-140, 50-130, 50-120, 50-110, 50-100, 50-90, 50-80, 50-70, 50-60, 60-150, 60-140, 60-130, 60-120, 60-110, 60-100, 60-90, 60-80, 60-70, 70-150, 70-140, 70-130, 70-120, 70-110, 70-100, 70-90, 70-80, 80-150. 80-140, 80-130, 80-120, 80-110, 80-100, 80-90, 90-150, 90-140, 90-130, 90-120, 90-110, 90-100, 100-150, 100-140, 100-130, 100-120, 100-110, 110-150, 110-140, 110-130, 110-120, 120-150, 120-140, 120-130, 130-150, 130-140, or 140-150. In some embodiments, the first solution comprises 10 mM sodium acetate, 7 mM acetic acid, and 100 mM NaCl, with a pH of 4.75. In some embodiments, the method further includes (d) washing the 3D structure from step (c) with a second solution, such as phosphate-buffered saline (PBS). In some embodiments, the method further includes drying the 3D structure from step (c) or step (d).

[0155] In some embodiments, the mass of therapeutic agent per cubic centimeter of structure in the device (e.g., a single therapeutic agent or a therapeutic agent attached to a target portion) is about 0.05 to 50 (mg / cc), for example, 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 number therein. For example, the therapeutic agent is approximately 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. A method for measuring the amount of a therapeutic peptide bound to a structure includes: (1) measuring the mass of the therapeutic peptide introduced into a first solution; (2) measuring the mass of the therapeutic agent remaining in the first solution after binding to and removing from the structure; (3) measuring the mass of the therapeutic agent in a second solution, if a washing step is included; (4) adding (2) and (3); and subtracting the sum of (4) from (1).

[0156] Treatment

[0157] On the other hand, methods for treating subjects using the structures described herein are provided. In some methods, the subject is treated with a device comprising a therapeutic peptide and a structure. In some cases, the subject suffers from a fracture or bone defect. In some cases, the subject requires vertebral fusion of the spine. In some cases, the subject suffers from cartilage tears or cartilage defects. In some cases, the subject suffers from cartilage loss.

[0158] In some embodiments, the subject has a defect in bone, cartilage, soft tissue, tendon, fascia, ligament, organ, bone tendon tissue, skin, or osteochondral, or a combination of one or more of the above defects. In some embodiments, the defect is the absence of bone, cartilage, soft tissue, tendon, fascia, ligament, organ, bone tendon tissue, skin, or osteochondral, or a combination of one or more of the above defects. In some embodiments, the defect in the subject is caused by trauma. In some embodiments, the defect in the subject is caused by a congenital condition. In some embodiments, the defect in the subject is caused by an acquired condition. In some embodiments, a defect refers to the absence, loss, and / or rupture of a tissue and / or organ of the body. In some embodiments, a "bone defect" refers to the absence or loss (e.g., partial loss) of bone at the subject's anatomical location, which should be present in a healthy control subject. Bone defects may result from infection (e.g., osteomyelitis), tumors, trauma, or adverse events related to treatment. Bone defects may also affect and damage the muscles, soft tissues, tendons, or joints surrounding the bone defect. In some embodiments, bone defects include damage to soft tissues. In some implementations, "cartilage defect" refers to the absence or loss (e.g., partial loss) of cartilage at an anatomical location in the subject, which should be present in a healthy control. Cartilage defects may result from disease, osteochondritis, osteonecrosis, or trauma. For example, cartilage defects may affect the knee joint.

[0159] Non-limiting examples of conditions suitable for treatment with the structures or devices described herein include osteoarthritis, intervertebral disc degeneration, congenital defects, spinal stenosis, vertebral displacement, vertebral stiffness, fractures, scoliosis, kyphosis, spinal fusion (PLF and intervertebral fusion), bone trauma repair, dental restoration, craniofacial restoration, ankle fusion, kyphosis correction, balloon osteotomy, scaphoid 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 combinations thereof. BMP2 is also involved in hair follicle development; therefore, this method may include treatment of hair follicles. Trauma can be bone, cartilage, soft tissue, tendon, fascia, ligament, organ, bone tendon tissue, or skin tissue or osteochondral tissue. In some embodiments, this method is used to treat osteochondral injuries.

[0160] This treatment method may include spinal fusion. In some embodiments, spinal fusion is a surgical technique that connects two or more vertebrae. In some embodiments, spinal fusion includes vertebral fusion (PLF). In some embodiments, spinal fusion includes intervertebral fusion.

[0161] This document provides methods for promoting bone or cartilage formation in a subject in need, methods comprising applying a therapeutically effective amount of any of the structures or devices described herein to the subject. Some embodiments of these methods may further include first selecting the subject in need of bone or cartilage formation. In some embodiments, the structure or device is applied to the vicinity of the site on the subject in which bone or cartilage formation is desired.

[0162] This document also provides methods for replacing and / or repairing bone or cartilage in a subject requiring such replacement, methods comprising applying a therapeutically effective amount of any of the structures or devices described herein to the subject. Some embodiments of these methods may further include first selecting the subject requiring bone replacement, bone repair, cartilage replacement, or cartilage repair. In some embodiments, the structure or device is applied near the site on the subject requiring bone or cartilage replacement or repair.

[0163] This document also provides methods for treating fractures or bone loss in a subject in need, methods comprising applying a therapeutically effective amount of any of the structures or devices described herein to the subject. Some embodiments of these methods may further include first selecting the subject suffering from a fracture or bone loss. In some embodiments, the structure or device is applied near the site of the fracture or bone loss in the subject.

[0164] This document also provides methods for repairing soft tissue in a subject requiring such repair, methods comprising applying a therapeutically effective amount of any of the structures or devices described herein to the subject. Some embodiments of these methods may further include first selecting a subject suffering from a fracture or bone loss. In some embodiments, the composition is applied to the vicinity of the site of the fracture or bone loss in the subject.

[0165] This document also provides methods for locally delivering a therapeutic agent to a subject in need, the method comprising applying to the subject any of the structures or devices described herein in a therapeutically effective amount. Some embodiments of these methods may further include first selecting a subject suffering from a fracture or bone loss. In some embodiments, the structure or device is applied near the site of the fracture or bone loss on the subject.

[0166] Methods for determining the therapeutic efficacy of a fracture or bone loss in a subject are known in the art and include, for example, imaging techniques (e.g., magnetic resonance imaging, X-ray, or computed tomography).

[0167] Methods for detecting bone or cartilage formation, or bone or cartilage replacement or repair, are also known in the art and include, for example, imaging techniques (e.g., magnetic resonance imaging, X-ray, or computed tomography).

[0168] Suitable animal models for the treatment of fractures or bone loss, bone or cartilage formation, or bone or cartilage replacement or repair are known in the art. Non-limiting examples of such animal models are described in the embodiments, 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.

[0169] As used herein, treatment methods include administering the structures or devices described herein to a subject. In some embodiments, administration includes a polypeptide or composition implanted herein.

[0170] In some embodiments, the polypeptide and / or composition comprising BMP-2 described herein is applied to a subject. In some embodiments, BMP2 comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with SEQ ID NO:454. In some embodiments, BMP-2 is applied to induce bone formation in a subject. In some embodiments, BMP-2 is applied to induce cartilage formation. In some embodiments, BMP-2 is applied in spinal fusion.

[0171] The terminology used herein is for the purpose of describing a particular situation only and is not intended to be limiting. Unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” are also intended to include the plural forms. With regard to the terms “comprising,” “having,” “with,” or variations thereof used in the detailed description and / or claims, these terms are intended to be inclusive in a manner similar to the term “comprising.”

[0172] In some implementations, the terms "about" or "approximately" refer to an acceptable margin of error for a particular value as determined by a person skilled in the art, which will depend in part on how the value is measured or determined, for example, the limitations of the measurement system. For example, "about" may refer to within one or more standard deviations based on practice for a given value.

[0173] As used herein, the term "object" refers to any mammal. Therefore, an object refers to, for example, a mouse, rat, dog, cat, horse, cow, pig, guinea pig, human, monkey, etc. When the object is a human, the object may be referred to herein as a patient. In some embodiments, the object or "object requiring treatment" may be a canine (e.g., a dog), a feline (e.g., a cat), an equine (e.g., a horse), a sheep, a bovine, a pig, a goat, a primate (e.g., an ape (e.g., a monkey (e.g., a marmoset, a baboon) or an ape (e.g., a gorilla, a chimpanzee, an orangutan, or a gibbon)), a human, or a rodent (e.g., a mouse, a guinea pig, a hamster, or a rat). In some embodiments, the object or "object requiring treatment" may be a non-human mammal, particularly a mammal commonly used as a model to demonstrate therapeutic efficacy in humans (e.g., a mouse, a rabbit, a pig, a canine, or a primate).

[0174] In some implementations, the term "therapeuticly effective amount" refers to the amount of a polypeptide or composition that effectively "treats" a disease, condition, or symptom. In some cases, a therapeutically effective amount of a polypeptide or composition reduces the severity of symptoms of a disease, condition, or symptom. In some cases, a disease, condition, or symptom includes organ or tissue defects.

[0175] 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 a polypeptide containing a β-TCP binding sequence) and its binding partner (e.g., β-TCP). Affinity can be measured by conventional methods known in the art, including those described herein. For example, affinity can be measured using surface plasmon resonance (SPR) techniques (e.g., ) or biological layer interferometry (e.g., To determine this.

[0176] The percentage of sequence identity (%) relative to a reference polypeptide sequence is the percentage of amino acid residues in the candidate sequence that are identical to those in the reference polypeptide sequence, after alignment and the introduction of gaps (if necessary) to obtain the maximum percentage of sequence identity, without considering any conserved substitutions as part of the sequence identity. Alignment for determining the percentage of amino acid sequence identity can be performed in various known ways, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Appropriate parameters for the aligned sequences can be determined, including the algorithms required to achieve maximum alignment on the full-length sequences to be compared. However, for the purposes of this paper, the sequence alignment computer program ALIGN-2 is used to generate the amino acid sequence identity % value. The ALIGN-2 sequence alignment computer program was written by Genentech, Inc., and the source code has been submitted with user documentation to the U.S. Copyright Office, Washington DC, 20559, registered under U.S. Copyright Registry No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or can be compiled from source code. The ALIGN-2 program should 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 will not be changed.

[0177] When performing amino acid sequence comparisons using ALIGN-2, the percentage of amino acid sequence identity between a given amino acid sequence A and a given amino acid sequence B (which can be alternatively expressed as a given amino acid sequence A having or containing a specific percentage of amino acid sequence identity with a given amino acid sequence B) is calculated as follows: 100 multiplied by the score X / Y, where X is the number of amino acid residues that are scored as identical matches by the sequence alignment program ALIGN-2 in the procedural alignment of A and B, and where Y is the total number of amino acid residues in B. It should be understood that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the percentage of amino acid sequence identity between A and B will not be equal to the percentage of amino acid sequence identity between B and A. Unless otherwise specified, all amino acid sequence identity percentage values ​​used herein were obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0178] Each of the embodiments described and illustrated herein has separate components and features that can be readily separated from or combined with features of any of the other embodiments without departing from the scope or spirit of the invention. Any enumerated method may be performed in the order of the enumerated events or in any other logically possible order.

[0179] Several embodiments of the present disclosure have been described. However, it should be understood that various changes may be made without departing from the spirit and scope of the present disclosure. Additionally, although specific formulations of the inks have been described, the specific amounts of each ink component may vary. Therefore, other embodiments are within the scope of the following claims.

[0180] Exemplary Implementation

[0181] 1. An apparatus comprising: a therapeutic agent and a printed three-dimensional structure comprising about 50% to about 90% by weight of ceramic and about 10% to about 50% by weight of polymer.

[0182] 2. The apparatus according to embodiment 1, wherein the therapeutic agent is non-covalently bound to the printed three-dimensional structure.

[0183] 3. The apparatus according to embodiment 1 or embodiment 2, wherein the ceramic comprises calcium phosphate, hydroxyapatite, fluorapatite, bone, silicate or vanadate, or a combination thereof.

[0184] 4. The apparatus according to any one of embodiments 1-3, wherein the ceramic comprises β-tricalcium phosphate (β-TCP).

[0185] 5. The device according to any one of embodiments 1-4, wherein the structure comprises about 70% to about 80% ceramic by weight.

[0186] 6. The apparatus according to embodiment 5, wherein the structure comprises about 75% ceramic by weight.

[0187] 7. The apparatus according to any one of embodiments 1-6, wherein the polymer comprises polyester.

[0188] 8. The apparatus according to any one of embodiments 1-7, wherein the polymer comprises polycaprolactone (PCL), polyglycolic acid, lactide, or polydioxanone (PDS), or a combination thereof.

[0189] 9. The apparatus according to any one of embodiments 1-8, wherein the polymer comprises polycaprolactone (PCL).

[0190] 10. The apparatus according to any one of embodiments 1-9, wherein the polymer comprises polyglycolic acid.

[0191] 11. The apparatus according to any one of embodiments 1-10, wherein the polymer comprises poly(p-dioxanone) (PDS).

[0192] 12. The apparatus according to any one of embodiments 1-11, wherein the polymer comprises lactide.

[0193] 13. The apparatus according to embodiment 12, wherein the lactide comprises L-lactide.

[0194] 14. The apparatus according to embodiment 12 or embodiment 13, wherein the lactide comprises poly(D,L-lactide).

[0195] 15. The apparatus according to any one of embodiments 1-14, wherein the polymer comprises a copolymer.

[0196] 16. The apparatus according to embodiment 15, wherein the copolymer comprises polycaprolactone (PCL) and polyglycolic acid.

[0197] 17. The apparatus according to embodiment 16, wherein the copolymer comprises about 90-95% PCL and about 5-10% polyethylene glycol based on molar weight.

[0198] 18. The apparatus according to any one of embodiments 15-17, wherein the copolymer comprises a poly(D,L-lactide-co-glycolic acid) copolymer.

[0199] 19. The apparatus according to embodiment 18, wherein the copolymer comprises about 40-60% poly(D,L-lactide) and about 40-60% polyglycolic acid (polyglycolic acid) on a molar basis.

[0200] 20. The apparatus according to any one of embodiments 15-19, wherein the copolymer comprises a PDS-glycolic acid copolymer.

[0201] 21. The apparatus according to embodiment 20, wherein the copolymer comprises about 90-95% PDS and about 5-10% glycolide on a molar basis.

[0202] 22. The apparatus according to any one of embodiments 15-21, wherein the copolymer comprises PDS-L-lactide copolymer.

[0203] 23. The apparatus according to embodiment 20, wherein the copolymer comprises about 90-95% PDS and about 5-10% L-lactide by molar weight.

[0204] 24. The apparatus according to any one of embodiments 1-23, wherein the structure comprises about 15% to about 25% by weight of a polymer.

[0205] 25. The apparatus according to embodiment 24, wherein the structure comprises about 25% polymer by weight.

[0206] 26. The apparatus according to any one of embodiments 1-25, wherein the printed three-dimensional structure is formed by ink comprising about 30% to about 70% by weight of ceramic, about 10% to about 30% by weight of polymer, and optionally comprising one or more additional reagents.

[0207] 27. The apparatus according to embodiment 26, wherein the ink comprises about 50% to about 70% ceramic by weight.

[0208] 28. The apparatus according to embodiment 27, wherein the ink comprises about 60% ceramic by weight.

[0209] 29. The apparatus according to any one of embodiments 26-28, wherein the ink comprises about 15% to about 25% ceramic by weight.

[0210] 30. The apparatus according to embodiment 29, wherein the ink comprises about 20% ceramic by weight.

[0211] 31. The apparatus according to any one of embodiments 26-30, wherein the ink contains one or more additional reagents.

[0212] 32. The apparatus according to any one of embodiments 26-31, wherein the ink contains one or more additional reagents by weight of about 1% to about 30%.

[0213] 33. The apparatus according to any one of embodiments 26-32, wherein one or more additional reagents include additional polymers, particles or foaming agents, or combinations thereof.

[0214] 34. The apparatus according to embodiment 33, wherein one or more additional reagents comprise additional polymers.

[0215] 35. The apparatus according to embodiment 33 or embodiment 34, wherein the additional polymer comprises a first polyethylene glycol and optionally a second polyethylene glycol, wherein if the second polyethylene glycol is present, its molecular weight is different from that of the first polyethylene glycol.

[0216] 36. The apparatus according to any one of embodiments 33-35, wherein the additional polymer is present in the ink at about 10% to about 30% by weight.

[0217] 37. The apparatus according to any one of embodiments 33-36, wherein one or more additional reagents comprise particles.

[0218] 38. The apparatus according to any one of embodiments 33-37, wherein the particles comprise sodium chloride, calcium chloride, sucrose, trehalose (e.g., α,α-trehalose dihydrate), or mannitol (e.g., D-mannitol), or combinations thereof.

[0219] 39. The apparatus according to any one of embodiments 33-38, wherein the particles are present in the ink at about 1% to about 10% by weight.

[0220] 40. The apparatus according to any one of embodiments 33-39, wherein one or more additional reagents include a foaming agent.

[0221] 41. The apparatus according to any one of embodiments 33-40, wherein the foaming agent comprises baking powder (e.g., monocalcium phosphate, sodium bicarbonate, corn starch) and / or azodicarbonamide.

[0222] 42. The apparatus according to any one of embodiments 33-41, wherein the particles are present in the ink at a weight of about 5% to about 20%.

[0223] 43. The device according to any one of embodiments 1-42, wherein the therapeutic agent comprises mammalian growth factor or a functional portion thereof.

[0224] 44. The device according to any one of embodiments 1-43, wherein the therapeutic agent comprises one or more polypeptides or functional portions thereof selected from Table 1.

[0225] 45. The device according to any one of embodiments 1-44, wherein the therapeutic agent comprises bone morphogenetic protein (BMP).

[0226] 46. ​​The apparatus according to any one of embodiments 1-45, wherein the therapeutic agent includes a targeting portion, and the targeting portion is non-covalently bound to the printed three-dimensional structure.

[0227] 47. The apparatus according to embodiment 46, wherein the targeting portion is bonded to the printed three-dimensional structure with an affinity of about 1 pM to about 100 μm.

[0228] 48. The apparatus according to embodiment 46 or embodiment 47, wherein the targeting portion comprises a polypeptide having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with any one of the sequences in Tables 2-3.

[0229] 49. The apparatus according to embodiment 46 or embodiment 47, wherein the targeting portion comprises about 2, 3, 4, 5, 6, 7, 8, 9 or 10 sequences selected from the sequences in Tables 2-3.

[0230] 50. The device according to any one of embodiments 1-49, wherein the therapeutic agent comprises a chimeric polypeptide comprising a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with any one of SEQ ID NO:433-441.

[0231] 51. A method for treating a condition in a subject, the method comprising administering to the subject the apparatus according to any one of embodiments 1-50.

[0232] 52. The method according to embodiment 51, wherein the condition includes bone defects, cartilage defects, soft tissue defects, tendon defects, fascia defects, ligament defects, organ defects, bone and tendon tissue defects, skin defects, osteochondral defects, osteoporosis, ischemic necrosis, or congenital skeletal deformities, or combinations thereof.

[0233] 53. The method according to embodiment 51 or embodiment 52, wherein the method includes spinal fusion.

[0234] 54. The method according to embodiment 53, wherein spinal fusion includes posterior lumbar fusion (PLF) and / or intervertebral fusion.

[0235] 55. The method according to embodiment 51 or embodiment 52, wherein the method includes bone repair, dental repair, craniofacial repair, ankle fusion, kyphosis correction, osteoplasty, scaphoid 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.

[0236] 56. A formulation comprising about 30% to about 70% by weight of ceramic, about 10% to about 30% by weight of polymer, and optionally comprising one or more additional reagents.

[0237] 57. The formulation according to embodiment 56, wherein the ceramic comprises calcium phosphate, hydroxyapatite, fluorapatite, bone, silicate or vanadate, or a combination thereof.

[0238] 58. The formulation according to embodiment 56 or embodiment 57, wherein the ceramic comprises β-tricalcium phosphate (β-TCP).

[0239] 59. The formulation according to any one of embodiments 56-58, wherein the ink comprises about 50% to about 70% ceramic by weight.

[0240] 60. The formulation according to embodiment 59, wherein the ink comprises about 60% ceramic by weight.

[0241] 61. The formulation according to any one of embodiments 56-60, wherein the polymer comprises polyester.

[0242] 62. The formulation according to any one of embodiments 56-61, wherein the polymer comprises polycaprolactone (PCL), polyglycolic acid, lactide, or polydioxanone (PDS), or a combination thereof.

[0243] 63. The formulation according to any one of embodiments 56-62, wherein the polymer comprises polycaprolactone (PCL).

[0244] 64. The formulation according to any one of embodiments 56-63, wherein the polymer comprises polyglycolic acid.

[0245] 65. The formulation according to any one of embodiments 56-64, wherein the polymer comprises poly(p-dioxanone) (PDS).

[0246] 66. The formulation according to any one of embodiments 56-65, wherein the polymer comprises lactide.

[0247] 67. The formulation according to any one of embodiments 56-66, wherein the lactide comprises L-lactide.

[0248] 68. The formulation according to any one of embodiments 56-67, wherein the lactide comprises poly(D,L-lactide).

[0249] 69. The formulation according to any one of embodiments 56-68, wherein the polymer comprises a copolymer.

[0250] 70. The formulation according to embodiment 69, wherein the copolymer comprises polycaprolactone (PCL) and polyglycolic acid.

[0251] 71. The formulation according to embodiment 70, wherein the copolymer comprises about 90-95% PCL and about 5-10% polyglycolic acid on a molar basis.

[0252] 72. The formulation according to any one of embodiments 69-71, wherein the copolymer comprises a poly(D,L-lactide-co-glycolic acid) copolymer.

[0253] 73. The formulation according to any one of embodiments 69-72, wherein the copolymer comprises about 40-60% poly(D,L-lactide) and about 40-60% polyglycolic acid (co-lactic acid) on a molar basis.

[0254] 74. The formulation according to any one of embodiments 69-73, wherein the copolymer comprises a PDS-glycolic acid copolymer.

[0255] 75. The formulation according to embodiment 74, wherein the copolymer comprises about 90-95% PDS and about 5-10% glycolide on a molar basis.

[0256] 76. The formulation according to any one of embodiments 69-75, wherein the copolymer comprises PDS-L-lactide copolymer.

[0257] 77. The formulation according to embodiment 76, wherein the copolymer comprises about 90-95% PDS and about 5-10% L-lactide on a molar basis.

[0258] 78. The formulation according to any one of embodiments 56-77, wherein the ink contains one or more additional reagents.

[0259] 79. The formulation according to any one of embodiments 56-78, wherein the ink contains about 1% to about 30% by weight of one or more additional reagents.

[0260] 80. The formulation according to any one of embodiments 56-79, wherein one or more additional agents comprise additional polymers, particles or foaming agents, or combinations thereof.

[0261] 81. The formulation according to embodiment 80, wherein one or more additional reagents comprise additional polymers.

[0262] 82. The formulation according to embodiment 80 or embodiment 81, wherein the additional polymer comprises a first polyethylene glycol and optionally a second polyethylene glycol, wherein if the second polyethylene glycol is present, its molecular weight is different from that of the first polyethylene glycol.

[0263] 83. The formulation according to any one of embodiments 80-82, wherein the additional polymer is present in the ink at about 10% to about 30% by weight.

[0264] 84. The formulation according to any one of embodiments 80-83, wherein one or more additional reagents comprise particles.

[0265] 85. The formulation according to any one of embodiments 80-84, wherein the particles comprise sodium chloride, calcium chloride, sucrose, trehalose (e.g., α,α-trehalose dihydrate) or mannitol (e.g., D-mannitol), or combinations thereof.

[0266] 86. The formulation according to any one of embodiments 80-85, wherein the particles are present in the ink at about 1% to about 10% by weight.

[0267] 87. The formulation according to any one of embodiments 80-86, wherein one or more additional reagents include a foaming agent.

[0268] 88. The formulation according to any one of embodiments 80-87, wherein the foaming agent comprises baking powder (e.g., monocalcium phosphate, sodium bicarbonate, corn starch) and / or azodicarbonamide.

[0269] 89. The formulation according to any one of embodiments 80-88, wherein the particles are present in the ink at a weight of about 5% to about 20%.

[0270] 90. A method for preparing a three-dimensional structure, the method comprising using a formulation according to any one of embodiments 56-89 in a three-dimensional printing method.

[0271] 91. A three-dimensional structure prepared using an ink formulation according to any one of embodiments 56-89.

[0272] 92. The structure according to embodiment 91, wherein the structure comprises about 50% to about 90% ceramic by weight.

[0273] 93. The structure according to embodiment 91, wherein the structure comprises about 50% to about 90% by weight of tricalcium phosphate.

[0274] 94. The structure according to any one of embodiments 91-93, wherein the structure comprises about 10% to about 50% by weight of a polymer.

[0275] 95. The structure according to embodiment 94, wherein the polymer comprises polyester.

[0276] 96. The structure according to embodiment 94 or embodiment 95, wherein the polymer comprises polycaprolactone (PCL), polyglycolic acid, lactide, or polydioxanone (PDS), or a combination thereof.

[0277] 97. The structure according to any one of embodiments 94-96, wherein the polymer comprises polycaprolactone (PCL).

[0278] 98. The structure according to any one of embodiments 94-97, wherein the polymer comprises polyglycolic acid.

[0279] 99. The structure according to any one of embodiments 94-98, wherein the polymer comprises poly(p-dioxanone) (PDS).

[0280] 100. The structure according to any one of embodiments 94-99, wherein the polymer comprises lactide.

[0281] 101. The structure according to embodiment 100, wherein the lactide comprises L-lactide.

[0282] 102. The structure according to embodiment 100 or embodiment 101, wherein the lactide comprises poly(D,L-lactide).

[0283] 103. The structure according to any one of embodiments 94-102, wherein the polymer comprises a copolymer.

[0284] 104. The structure according to embodiment 103, wherein the copolymer comprises polycaprolactone (PCL) and polyglycolic acid.

[0285] 105. The structure according to embodiment 104, wherein the copolymer comprises about 90-95% PCL and about 5-10% polyglycolic acid based on molar weight.

[0286] 106. The structure according to any one of embodiments 103-105, wherein the copolymer comprises a poly(D,L-lactide-co-glycolic acid) copolymer.

[0287] 107. The structure according to any one of embodiments 103-106, wherein the copolymer comprises about 40-60% poly(D,L-lactide) and about 40-60% polyglycolic acid based on molar weight.

[0288] 108. The structure according to any one of embodiments 103-107, wherein the copolymer comprises a PDS-glycolic acid copolymer.

[0289] 109. The structure according to embodiment 108, wherein the copolymer comprises about 90-95% PDS and about 5-10% glycolide on a molar basis.

[0290] 110. The structure according to any one of embodiments 103-109, wherein the copolymer comprises a PDS-L-lactide copolymer.

[0291] 111. The structure according to embodiment 110, wherein the copolymer comprises about 90-95% PDS and about 5-10% L-lactide on a molar basis.

[0292] 112. The structure according to any one of embodiments 94-111, wherein the structure comprises about 15% to about 25% of a polymer by weight.

[0293] 113. The structure according to embodiment 112, wherein the structure comprises about 25% polymer by weight.

[0294] 114. A three-dimensional structure comprising about 50% to about 90% by weight of ceramic and about 10% to about 30% by weight of polymer.

[0295] 115. The structure according to embodiment 114, wherein the ceramic comprises calcium phosphate, hydroxyapatite, fluorapatite, bone, silicate or vanadate, or a combination thereof.

[0296] 116. The structure according to embodiment 114 or embodiment 115, wherein the ceramic comprises β-tricalcium phosphate (β-TCP).

[0297] 117. The structure according to any one of embodiments 114-116, wherein the structure comprises about 65% to about 85% ceramic by weight.

[0298] 118. The structure according to embodiment 117, wherein the structure comprises about 70% to about 80% ceramic by weight.

[0299] 119. The structure according to embodiment 118, wherein the structure comprises about 75% ceramic by weight.

[0300] 120. The structure according to any one of embodiments 114-119, wherein the polymer comprises polyester.

[0301] 121. The structure according to any one of embodiments 114-120, wherein the polymer comprises polycaprolactone (PCL), polyglycolic acid, lactide, or polydioxanone (PDS), or a combination thereof.

[0302] 122. The structure according to any one of embodiments 114-121, wherein the polymer comprises polycaprolactone (PCL).

[0303] 123. The structure according to any one of embodiments 114-122, wherein the polymer comprises polyglycolic acid.

[0304] 124. The structure according to any one of embodiments 114-123, wherein the polymer comprises poly(p-dioxanone) (PDS).

[0305] 125. The structure according to any one of embodiments 114-124, wherein the polymer comprises lactide.

[0306] 126. The structure according to embodiment 125, wherein the lactide comprises L-lactide.

[0307] 127. The structure according to embodiment 125 or embodiment 126, wherein the lactide comprises poly(D,L-lactide).

[0308] 128. The structure according to any one of embodiments 114-127, wherein the polymer comprises a copolymer.

[0309] 129. The structure according to embodiment 128, wherein the copolymer comprises polycaprolactone (PCL) and polyglycolic acid.

[0310] 130. The structure according to embodiment 129, wherein the copolymer comprises about 90-95% PCL and about 5-10% polyglycolic acid based on molar weight.

[0311] 131. The structure according to any one of embodiments 128-130, wherein the copolymer comprises a poly(D,L-lactide-co-glycolic acid) copolymer.

[0312] 132. The structure according to embodiment 131, wherein the copolymer comprises about 40-60% poly(D,L-lactide) and about 40-60% polyglycolic acid (co-lactic acid) on a molar basis.

[0313] 133. The structure according to any one of embodiments 128-132, wherein the copolymer comprises a PDS-glycolic acid copolymer.

[0314] 134. The structure according to embodiment 133, wherein the copolymer comprises about 90-95% PDS and about 5-10% glycolide on a molar basis.

[0315] 135. The structure according to any one of embodiments 128-134, wherein the copolymer comprises a PDS-L-lactide copolymer.

[0316] 136. The structure according to embodiment 135, wherein the copolymer comprises about 90-95% PDS and about 5-10% L-lactide on a molar basis.

[0317] 137. The structure according to any one of embodiments 114-136, wherein the structure comprises about 15% to about 25% of a polymer by weight.

[0318] 138. The structure according to embodiment 137, wherein the structure comprises about 25% polymer by weight.

[0319] 139. The structure according to any one of embodiments 91-138, wherein the structure is prepared by a three-dimensional printing method.

[0320] 140. A method for manufacturing a three-dimensional structure according to any one of embodiments 91-139, the method comprising depositing an ink formulation in a three-dimensional form.

[0321] 141. The method according to embodiment 140, wherein the ink formulation comprises the ink formulation according to any one of embodiments 56-89.

[0322] 142. A method for treating a condition of a subject in need, the method comprising delivering to an organ or tissue of the subject a structure according to any one of embodiments 91-139.

[0323] 143. A method for treating a condition of a subject in need, the method comprising delivering to an organ or tissue of the subject a structure manufactured by the method according to any one of embodiments 90, 140 or 141.

[0324] 144. The method according to embodiment 142 or embodiment 143, wherein the condition includes bone defects, cartilage defects, soft tissue defects, tendon defects, fascia defects, ligament defects, organ defects, bone and tendon tissue defects, skin defects, osteochondral defects, osteoporosis, ischemic necrosis or congenital skeletal deformities, or combinations thereof.

[0325] 145. The method according to any one of embodiments 142-144, wherein the method includes spinal fusion.

[0326] 146. The method according to embodiment 145, wherein spinal fusion includes posterior lumbar fusion (PLF) and / or intervertebral fusion.

[0327] 147. The method according to embodiment 142 or embodiment 143, wherein the method includes bone repair, dental repair, craniofacial repair, ankle fusion, kyphosis correction, osteoplasty, scaphoid 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.

[0328] 148. The method according to any one of embodiments 142-147, further comprising treating the subject with a therapeutic agent.

[0329] 149. A method for delivering a therapeutic agent to a subject in need, the method comprising delivering to an organ or tissue of the subject an apparatus containing the therapeutic agent and a structure according to any one of embodiments 91-139.

[0330] 150. The method according to embodiment 148 or embodiment 149, wherein the therapeutic agent comprises a mammalian growth factor or a functional portion thereof.

[0331] 151. The method according to any one of embodiments 148-150, wherein the therapeutic agent comprises one or more polypeptides or functional portions thereof selected from Table 1.

[0332] 152. The method according to any one of embodiments 148-151, wherein the therapeutic agent comprises bone morphogenetic protein (BMP).

[0333] 153. The method according to any one of embodiments 148-152, wherein the therapeutic agent comprises a target portion of the structure that is non-covalently bound to it.

[0334] 154. The method according to embodiment 153, wherein the targeting portion binds to the printed three-dimensional structure with an affinity of about 1 pM to about 100 μm.

[0335] 155. The method according to embodiment 153 or embodiment 154, wherein the targeted portion comprises a polypeptide having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with any of the sequences in Tables 5-6.

[0336] 156. The method according to embodiment 153 or embodiment 154, wherein the targeting portion comprises about 2, 3, 4, 5, 6, 7, 8, 9 or 10 sequences selected from the sequences in Tables 5-6.

[0337] 157. The method according to any one of embodiments 148-156, wherein the therapeutic agent comprises a chimeric polypeptide or a portion thereof, the chimeric polypeptide comprising a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with any one of SEQ ID NO:433-441.

[0338] 158. The apparatus according to any one of embodiments 1-50, the method according to any one of embodiments 51-55, 90, 140-157, or the structure according to any one of embodiments 91-139, wherein the density of the structure is about 1 g / cm³. 3 Approximately 2g / cm 3 .

[0339] 159. The apparatus according to any one of embodiments 1-50, the method according to any one of embodiments 51-55, 90, 140-157, or the structure according to any one of embodiments 91-139, wherein the open porosity of the structure is about 15% to about 50%.

[0340] 160. The apparatus according to any one of embodiments 1-50, the method according to any one of embodiments 51-55, 90, 140-157, or the structure according to any one of embodiments 91-139, wherein the diameter of the support column of the structure is about 300 μm to about 600 μm.

[0341] 161. A filament comprising the formulation according to any one of embodiments 56-89.

[0342] 162. The filament according to embodiment 161, wherein the diameter of the filament is about 1.5 mm to about 2 mm.

[0343] 163. A granule comprising the formulation according to any one of embodiments 56-89.

[0344] 164. The pellets according to embodiment 163, wherein the length of the pellets is about 3 mm to about 4 mm.

[0345] Example

[0346] Example 1: Ink formulation and 3D printed scaffold

[0347] Ink formulation and scaffold #1:

[0348] The 3D printing ink material is a flexible polymer-ceramic composite containing β-TCP, which can embolize therapeutic agents such as tBMP2. The formulation contains two sacrificial pore-forming agents (water-soluble polyethylene glycol and water-soluble sucrose) to expose more β-TCP surface area for tBMP2 binding. The 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 is immersed in water to dissolve the sacrificial polyethylene glycol and sucrose pore-forming agents. The resulting scaffold #1 contains 75% β-TCP powder and 25% polycaprolactone by weight.

[0349] The ink was also converted into 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 was immersed in water to dissolve the sacrificial polyethylene glycol and sucrose pore-forming agent. The resulting scaffold contained 75% by weight β-TCP powder and 25% by weight polycaprolactone.

[0350] The ink is also converted into granules (e.g., filaments are formed into small granules) and fed from the hopper into a micro-screw extruder, which melts the material and pushes it out of the fine nozzle.

[0351] The ink was also ground into a fine powder at low temperature. The powder was then laser-heated into the desired configuration using the SLS method.

[0352] Table 4. Exemplary Ink Formulation #1

[0353]

[0354]

[0355] Ink Formulation #2

[0356] The 3D printing ink material is a flexible polymer-ceramic composite containing β-TCP, which can embolize therapeutic agents such as tBMP2. The formulation contains a sacrificial pore-forming agent (water-soluble polyethylene glycol) to expose more β-TCP surface area for agent binding. The formulation uses a 95 mol% caprolactone and 5 mol% glycolide copolymer, which exhibits faster bioabsorption properties compared to polycaprolactone. The 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 is immersed in water to dissolve the sacrificial polyethylene glycol pore-forming agent. The resulting scaffold #2 contains 75% by weight β-TCP powder and 25% by weight caprolactone / glycolic acid copolymer (95:5).

[0357] The ink was also converted into 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 was immersed in water to dissolve the sacrificial polyethylene glycol pore-forming agent. The resulting scaffold contained 75% by weight β-TCP powder and 25% by weight caprolactone / glycolic acid copolymer (95:5).

[0358] The ink is also converted into granules (e.g., filaments are formed into small granules) and fed from the hopper into a micro-screw extruder, which melts the material and pushes it out of the fine nozzle.

[0359] The ink was also ground into a fine powder at low temperature. The powder was then laser-heated into the desired configuration using the SLS method.

[0360] Table 5. Exemplary Ink Formulation #2

[0361]

[0362] Ink Formulation #3

[0363] The 3D printing ink material is a flexible polymer-ceramic composite containing β-TCP, which can embolize therapeutic agents such as tBMP2. The formulation contains a sacrificial pore-forming agent (water-soluble polyethylene glycol) to expose more β-TCP surface area for agent binding. The formulation uses a 90 mol% caprolactone and 10 mol% glycolide copolymer, which exhibits rapid bioabsorption characteristics compared to polycaprolactone. The 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 is immersed in water to dissolve the sacrificial polyethylene glycol pore-forming agent. The resulting scaffold #3 contains 75% by weight β-TCP powder and 25% by weight caprolactone / glycolic acid copolymer (90:10).

[0364] The ink was also converted into 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 was immersed in water to dissolve the sacrificial polyethylene glycol pore-forming agent. The resulting scaffold contained 75% by weight β-TCP powder and 25% by weight caprolactone / glycolic acid copolymer (90:10).

[0365] The ink is also converted into granules (e.g., filaments are formed into small granules) and fed from the hopper into a micro-screw extruder, which melts the material and pushes it out of the fine nozzle.

[0366] The ink was also ground into a fine powder at low temperature. The powder was then laser-heated into the desired configuration using the SLS method.

[0367] Table 6. Exemplary Ink Formulation #3

[0368]

[0369] Ink Formulation #4

[0370] The 3D printing ink material is a flexible polymer-ceramic composite containing β-TCP, which can embolize therapeutic agents such as tBMP2. The formulation contains a sacrificial pore-forming agent (water-soluble polyethylene glycol) to expose more β-TCP surface area for agent binding. The formulation uses a 50 mol%:50 mol% poly(D,L-lactide-co-glycolic acid) copolymer, which has rapid bioabsorption properties compared to polycaprolactone. The 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 is immersed in water to dissolve the sacrificial polyethylene glycol pore-forming agent. The resulting scaffold #4 contains 75% by weight β-TCP powder and 25% by weight poly(D,L-lactide-co-glycolic acid) copolymer (50:50).

[0371] The ink was also converted into 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 was immersed in water to dissolve the sacrificial polyethylene glycol pore-forming agent. The resulting scaffold contained 75% by weight β-TCP powder and 25% by weight poly(D,L-lactide-co-glycolic acid) copolymer (50:50).

[0372] The ink is also converted into granules (e.g., filaments are formed into small granules) and fed from the hopper into a micro-screw extruder, which melts the material and pushes it out of the fine nozzle.

[0373] The ink was also ground into a fine powder at low temperature. The powder was then laser-heated into the desired configuration using the SLS method.

[0374] Table 7. Exemplary Ink Formulation #4

[0375]

[0376] Ink Formulation #5

[0377] The 3D printing ink material is a flexible polymer-ceramic composite containing β-TCP, which can be embolized to therapeutic agents such as tBMP2. The formulation contains a sacrificial pore-forming agent (water-soluble polyethylene glycol) to expose more β-TCP surface area for agent binding. The ink is a medium-viscosity formulation that forms 1.75 mm filaments for 3D printing on RepRap-type FFF 3D printers with nozzles of 400 μm diameter (e.g., the Prusa i3 MK3S 3D printer). Higher molecular weight polyethylene glycol (8000 MW for FFF 3D printing and 1500 MW for syringe-based bioprinting) produces a higher viscosity material, which facilitates the extrusion of the 1.75 mm diameter filament. After 3D printing, the resulting scaffold is immersed in water to dissolve the sacrificial polyethylene glycol pore-forming agent. The resulting scaffold #5 contains 75% β-TCP powder and 25% polycaprolactone by weight.

[0378] The ink is also converted into granules (e.g., filaments are formed into small granules) and fed from the hopper into a micro-screw extruder, which melts the material and pushes it out of the fine nozzle.

[0379] The ink was also ground into a fine powder at low temperature. The powder was then laser-heated into the desired configuration using the SLS method.

[0380] Table 8. Exemplary Ink Formulation #5

[0381]

[0382] Ink Formulation #6

[0383] This 3D printing ink material is a flexible polymer-ceramic composite containing β-TCP, which can be embolized to therapeutic agents such as tBMP2. The formulation contains a sacrificial pore-forming agent (water-soluble polyethylene glycol) to expose more β-TCP surface area for agent binding. The ink also contains a foaming agent (sodium bicarbonate), which thermally decomposes during 3D printing and releases CO2 gas to create a foam structure, thereby increasing the porosity of the 3D-printed scaffold. The ink is a medium-viscosity formulation that forms 1.75 mm filaments for 3D printing on RepRap-type FFF 3D printers with nozzles of 400 μm diameter (e.g., the Prusa i3 MK3S 3D printer). Higher molecular weight polyethylene glycol (8000 MW for FFF 3D printing and 1500 MW for syringe-based bioprinting) produces a higher viscosity material, which facilitates 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 and sodium carbonate byproducts from the thermal decomposition of sodium bicarbonate. The resulting scaffold #6 contained 75% β-TCP powder and 25% polycaprolactone by weight.

[0384] The ink is also converted into granules (e.g., filaments are formed into small granules) and fed from the hopper into a micro-screw extruder, which melts the material and pushes it out of the fine nozzle.

[0385] The ink was also ground into a fine powder at low temperature. The powder was then laser-heated into the desired configuration using the SLS method.

[0386] Table 9. Exemplary Ink Formulation #6

[0387]

[0388] Ink Formulation #7

[0389] The 3D printing ink material is a flexible polymer-ceramic composite containing β-TCP, which can embolize therapeutic agents such as tBMP2. The formulation contains a sacrificial pore-forming agent (water-soluble polyethylene glycol) to expose more β-TCP surface area for agent binding. The formulation uses a 90 mol%:10 mol% poly(p-dioxanone-co-L-lactide) copolymer, which has faster bioabsorption properties compared to polycaprolactone. The 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% by weight β-TC powder and 25% by weight p-dioxanone / L-lactide copolymer (90:10).

[0390] The ink was also converted into 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 was immersed in water to dissolve the sacrificial polyethylene glycol pore-forming agent. The resulting scaffold contained 75% by weight β-TC powder and 25% by weight p-dioxanone / L-lactide copolymer (90:10).

[0391] The ink is also converted into granules (e.g., filaments are formed into small granules) and fed from the hopper into a micro-screw extruder, which melts the material and pushes it out of the fine nozzle.

[0392] The ink was also ground into a fine powder at low temperature. The powder was then laser-heated into the desired configuration using the SLS method.

[0393] Table 10. Exemplary Ink Formulation #7

[0394]

[0395]

[0396] Ink Formulation #8

[0397] The 3D printing ink material is a flexible polymer-ceramic composite containing β-TCP, which can be embolized to therapeutic agents such as tBMP2. The formulation contains a sacrificial pore-forming agent (water-soluble polyethylene glycol) to expose more β-TCP surface area for agent binding. The ink is a medium-viscosity formulation that can form 1.75 mm filaments for 3D printing on RepRap-type FFF 3D printers with nozzles of 400 μm diameter (e.g., the Prusa i3 MK3S 3D printer). Higher molecular weight polyethylene glycol blends (8000MW and 20000MW for FFF 3D printing, and 1500MW for syringe-based bioprinting) produce a higher viscosity material, which facilitates the extrusion of 1.75 mm diameter filaments. After 3D printing is complete, the resulting scaffold is immersed in water to dissolve the sacrificial polyethylene glycol pore-forming agent. The resulting scaffold contains 75% by weight β-TCP powder and 25% by weight polycaprolactone.

[0398] The ink is also converted into granules (e.g., filaments are formed into small granules) and fed from the hopper into a micro-screw extruder, which melts the material and pushes it out of the fine nozzle.

[0399] The ink was also ground into a fine powder at low temperature. The powder was then laser-heated into the desired configuration using the SLS method.

[0400] Table 11. Exemplary Ink Formulation #8

[0401]

[0402]

[0403] Ink Formulation #9

[0404] This 3D printing ink material is a flexible polymer-ceramic composite containing β-TCP, which can be embolized to therapeutic agents such as tBMP2. The formulation contains two sacrificial pore-forming agents (water-soluble polyethylene glycol and water-soluble glucose) to expose more β-TCP surface area for agent binding. The ink is a medium-viscosity formulation that can form 1.75 mm filaments for 3D printing on RepRap-type FFF 3D printers with nozzles of 400 μm diameter (e.g., the Prusa i3 MK3S 3D printer). Higher molecular weight polyethylene glycol blends (8000MW and 20000MW for FFF 3D printing, and 1500MW for syringe-based bioprinting) produce a higher viscosity material, which facilitates the extrusion of 1.75 mm diameter filaments. After 3D printing is complete, the resulting scaffold is immersed in water to dissolve the sacrificial polyethylene glycol and sucrose pore-forming agents. The resulting scaffold contains 75% by weight β-TCP powder and 25% by weight polycaprolactone.

[0405] The ink is also converted into granules (e.g., filaments are formed into small granules) and fed from the hopper into a micro-screw extruder, which melts the material and pushes it out of the fine nozzle.

[0406] The ink was also ground into a fine powder at low temperature. The powder was then laser-heated into the desired configuration using the SLS method.

[0407] Table 12. Exemplary Ink Formulation #9

[0408]

[0409]

[0410] Ink Formulation #10

[0411] This 3D printing ink material is a flexible polymer-ceramic composite containing β-TCP, which can be embolized to therapeutic agents such as tBMP2. The formulation contains a sacrificial pore-forming agent (water-soluble polyethylene glycol) to expose more β-TCP surface area for agent binding. The formulation uses a 95 mol% caprolactone and 5 mol% glycolide copolymer, which exhibits faster bioabsorption properties compared to polycaprolactone. This ink is a medium-viscosity formulation that can form 1.75 mm filaments for 3D printing on RepRap-type FFF 3D printers with 400 μm diameter nozzles (e.g., the Prusa i3 MK3S 3D printer). Higher molecular weight polyethylene glycol blends (8000MW and 20000MW for FFF 3D printing, and 1500MW for syringe-based bioprinting) produce a higher viscosity material, which facilitates 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 contained 75% by weight β-TCP powder and 25% by weight caprolactone / glycolic acid copolymer (95:5).

[0412] The ink is also converted into granules (e.g., filaments are formed into small granules) and fed from the hopper into a micro-screw extruder, which melts the material and pushes it out of the fine nozzle.

[0413] The ink was also ground into a fine powder at low temperature. The powder was then laser-heated into the desired configuration using the SLS method.

[0414] Table 13. Exemplary Ink Formulation #10

[0415]

[0416] Ink Formulation #11

[0417] This 3D printing ink material is a flexible polymer-ceramic composite containing β-TCP, which can be embolized to therapeutic agents such as tBMP2. The formulation contains a sacrificial pore-forming agent (water-soluble polyethylene glycol) to expose more β-TCP surface area for agent binding. The formulation uses a 90 mol% caprolactone and 10 mol% glycolide copolymer, which exhibits rapid bioabsorption characteristics compared to polycaprolactone. This ink is a medium-viscosity formulation that can form 1.75 mm filaments for 3D printing on RepRap-type FFF 3D printers with 400 μm diameter nozzles (e.g., the Prusa i3 MK3S 3D printer). Higher molecular weight polyethylene glycol blends (8000MW and 20000MW for FFF 3D printing, and 1500MW for syringe-based bioprinting) produce a higher viscosity material, which facilitates 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 contained 75% by weight β-TCP powder and 25% by weight caprolactone / glycolic acid copolymer (90:10).

[0418] The ink is also converted into granules (e.g., filaments are formed into small granules) and fed from the hopper into a micro-screw extruder, which melts the material and pushes it out of the fine nozzle.

[0419] The ink was also ground into a fine powder at low temperature. The powder was then laser-heated into the desired configuration using the SLS method.

[0420] Table 14. Exemplary Ink Formulation #11

[0421]

[0422] Ink Formulation #12

[0423] This 3D printing ink material is a flexible polymer-ceramic composite containing β-TCP, which can be embolized to therapeutic agents such as tBMP2. The formulation contains a sacrificial pore-forming agent (water-soluble polyethylene glycol) to expose more β-TCP surface area for agent binding. The formulation uses a 50 mol%:50 mol% poly(D,L-lactide-co-glycolic acid) copolymer, which exhibits rapid bioabsorption characteristics compared to polycaprolactone. This ink is a medium-viscosity formulation that can form 1.75 mm filaments for 3D printing on RepRap-type FFF 3D printers with nozzles of 400 μm diameter (e.g., the Prusa i3 MK3S 3D printer). Higher molecular weight polyethylene glycol blends (8000MW and 20000MW for FFF 3D printing, and 1500MW for syringe-based bioprinting) produce a higher viscosity material, which facilitates 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 contained 75% by weight β-TCP powder and 25% by weight poly(D,L-lactide-co-glycolic acid) copolymer (50:50).

[0424] The ink is also converted into granules (e.g., filaments are formed into small granules) and fed from the hopper into a micro-screw extruder, which melts the material and pushes it out of the fine nozzle.

[0425] The ink was also ground into a fine powder at low temperature. The powder was then laser-heated into the desired configuration using the SLS method.

[0426] Table 15. Exemplary Ink Formulation #12

[0427]

[0428]

[0429] Ink Formulation #13

[0430] The 3D printing ink material is a flexible polymer-ceramic composite containing β-TCP, which can be embolized to therapeutic agents such as tBMP2. The formulation contains a sacrificial pore-forming agent (water-soluble polyethylene glycol) to expose more β-TCP surface area for agent 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.

[0431] The ink is also converted into granules (e.g., filaments are formed into small granules) and fed from the hopper into a micro-screw extruder, which melts the material and pushes it out of the fine nozzle.

[0432] The ink was also ground into a fine powder at low temperature. The powder was then laser-heated into the desired configuration using the SLS method.

[0433] Table 16. Exemplary Ink Formulation #13

[0434] β-TCP powder 50-80 Poly(p-dioxanone) (PDS) 10-30 polyethylene glycol 10-30

[0435] Ink Formulation #14

[0436] The 3D printing ink material is a flexible polymer-ceramic composite containing β-TCP, which can be embolized to therapeutic agents such as tBMP2. The formulation contains a sacrificial pore-forming agent (water-soluble polyethylene glycol) to expose more β-TCP surface area for agent 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-glycolic acid copolymer (90:10).

[0437] The ink is also converted into granules (e.g., filaments are formed into small granules) and fed from the hopper into a micro-screw extruder, which melts the material and pushes it out of the fine nozzle.

[0438] The ink was also ground into a fine powder at low temperature. The powder was then laser-heated into the desired configuration using the SLS method.

[0439] Table 17. Exemplary Ink Formulation #14

[0440] β-TCP powder 50-80 PDS-glycolic acid copolymer (90:10) 10-30 polyethylene glycol 10-30

[0441] Ink Formulation #15

[0442] The 3D printing ink material is a flexible polymer-ceramic composite containing β-TCP, which can be embolized to therapeutic agents such as tBMP2. The formulation contains a sacrificial pore-forming agent (water-soluble polyethylene glycol) to expose more β-TCP surface area for agent 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 L-lactide copolymer (90:10).

[0443] The ink is also converted into granules (e.g., filaments are formed into small granules) and fed from the hopper into a micro-screw extruder, which melts the material and pushes it out of the fine nozzle.

[0444] The ink was also ground into a fine powder at low temperature. The powder was then laser-heated into the desired configuration using the SLS method.

[0445] Table 18. Exemplary Ink Formulation #15

[0446] β-TCP powder 50-80 PDS-L-lactide copolymer (90:10) 10-30 polyethylene glycol 10-30

[0447] Example 2: Ink preparation and scaffold fabricated by 3D printing

[0448] Ink formulations and scaffolds #1

[0449] Methods: To prepare 5.3cc batches 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 container. The wide-mouthed glass flask was placed in a FlackTek speedmixer and mixed at low intensity (300 rpm) for 2 min to homogenize the powder blend before high-rpm mixing. The mixer was then set to high intensity (3500 rpm) for 5 min. During mixing, internal friction caused the polycaprolactone and polyethylene glycol to melt, transforming the ink into a viscous molten liquid. This liquid-phase mixing promoted the tight dispersion of the β-TCP and sucrose powders into the molten polymer blend. The ink blend was cooled for 10–15 min and then mixed again at 3500 rpm for 5 min. The mixing / cooling process was repeated for a total of four 5-min mixing sessions at 3500 rpm. After the fourth mixing at 3500 rpm, pour the ink feed onto a glass plate and use two scrapers to form a cylinder approximately 1 cm in diameter and 6 cm long. While the ink is still semi-molten, cut it into several slices approximately 1-2 cm long using a straight razor.

[0450] 3D Printing: Transfer the solid polymer / β-TCP sheet into a 5cc stainless steel syringe (for an Allevi 3 bioprinter). Heat the extruder CORE printhead to 135°C and hold it for approximately 30 minutes to ensure the ink melts. Print the ink using a 400-micron inner diameter conical metal Luer head, at 70 psi pressure and a nozzle speed of 7 mm / s. 3D print the scaffold onto a piece of craft tape applied to a smooth glass or polymer surface (such as a glass microscope slide, a large glass plate, or a 96-well plate cover).

[0451] Post-processing: The 3D-printed structure was soaked overnight in distilled water to dissolve the polyethylene glycol and sucrose in the printing material, resulting in a porous and flexible β-TCP / polycaprolactone composite. The scaffold was dried for at least 12 hours to ensure that any residual water had evaporated from the porous scaffold before binding with therapeutic agents such as tBMP2 protein. The scaffold was sterilized by soaking in a 70% ethanol solution for 2–4 hours and then dried in a biosafety cabinet for approximately 12 hours. Images of the scaffold are shown below. Figures 1A-1C .

[0452] 3D printing was also performed using an FFF 3D printer. Ink #1 was extruded into a filament, which was then loaded into the FFF 3D printer to create a 3D-printed scaffold. The scaffold was then processed using the post-processing methods described above.

[0453] Ink formulations and scaffolds #2

[0454] Methods: To prepare 5cc batches of ink, 5.6 g of β-TCP powder, 1.87 g of 95:5 caprolactone / glycolic acid copolymer granules, and 1.87 g of polyethylene glycol flakes were added to a glass mixing container. The wide-mouth glass flask was placed in a FlackTek speedmixer and mixed at low intensity (300 rpm) for 2 min to homogenize the powder blend before high-rpm mixing. The mixture was then mixed at high intensity (3500 rpm) for 5 min. During mixing, internal friction caused the 95:5 caprolactone / glycolic acid copolymer and polyethylene glycol to melt, turning the ink into a viscous molten liquid. This liquid-phase mixing promoted the tight dispersion of the β-TCP powder into the molten polymer blend. The blended ink was cooled for 10–15 min and then mixed again at 3500 rpm for 5 min. The mixing / cooling process was repeated for a total of four 5-min mixing sessions at 3500 rpm. After the fourth mixing at 3500 rpm, pour the ink feed onto a glass plate and use two scrapers to form a cylinder approximately 1 cm in diameter and 6 cm long. While the ink is still semi-molten, cut it into several slices approximately 1-2 cm long using a straight razor.

[0455] 3D Printing: Transfer the solid polymer / β-TCP sheet into a 5cc stainless steel syringe (for an Allevi 3 bioprinter). Heat the extruder CORE printhead to 130°C and hold for approximately 30 minutes to ensure the ink melts. Print the ink using a 320-micron inner diameter conical metal Luer head, applying 80 psi pressure and a nozzle speed of 6 mm / s. 3D print the scaffold onto a piece of craft tape applied to a smooth glass or polymer surface (such as a glass microscope slide, a large glass plate, or a 96-well plate cover).

[0456] Post-processing: The 3D-printed structure was soaked overnight in distilled water to dissolve the polyethylene glycol in the printing material, resulting in a porous and flexible β-TCP / 95:5 caprolactone / glycolic acid copolymer composite. The scaffold was dried for at least 12 hours to ensure that any residual water evaporated from the porous scaffold before binding with a therapeutic agent (such as tBMP2). The scaffold was sterilized by soaking in a 70% ethanol solution for 2–4 hours and then dried in a biosafety cabinet for approximately 12 hours. Images of the scaffold are shown below. Figures 2A-2C .

[0457] 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 create a 3D-printed scaffold. The scaffold was then processed using the post-processing methods described above.

[0458] Ink formulations and scaffolds #3

[0459] Method: To prepare 5cc batches of ink, 5.6 g of β-TCP powder, 1.87 g of 90:10 caprolactone / glycolic acid copolymer fragments, and 1.87 g of polyethylene glycol flakes were added to a glass mixing container. The mixture was placed in a wide-mouth glass flask of a dual asymmetric centrifugal mixer (FlackTek Speedmixer) and mixed at low intensity (300 rpm) for 2 min to homogenize the powder blend before high-rpm mixing. It was then mixed at high intensity (3500 rpm) for 5 min. During mixing, internal friction caused the 90:10 caprolactone / glycolic acid copolymer and polyethylene glycol to melt, turning the ink into a viscous molten liquid. This liquid-phase mixing promoted the tight dispersion of the β-TCP powder into the molten polymer blend. The blended ink was cooled for 10–15 min and then mixed again at 3500 rpm for 5 min. The mixing / cooling process was repeated for a total of four 5-min mixing sessions at 3500 rpm. After the fourth mixing at 3500 rpm, pour the ink feed onto a glass plate and use two scrapers to form a cylinder approximately 1 cm in diameter and 6 cm long. While the ink is still semi-molten, cut it into several slices approximately 1-2 cm long using a straight razor.

[0460] 3D Printing: Transfer the solid polymer / β-TCP sheet into a 5cc stainless steel syringe (for an Allevi 3 bioprinter). Heat the extruder CORE printhead to 130°C and hold for approximately 30 minutes to ensure the ink melts. Print the ink using a 320-micron inner diameter conical metal Luer head, applying 45 psi pressure and a nozzle speed of 7 mm / s. 3D print the scaffold onto a piece of craft tape applied to a smooth glass or polymer surface (such as a glass microscope slide, a large glass plate, or a 96-well plate cover).

[0461] Post-processing: The 3D-printed structure was soaked overnight in distilled water to dissolve the polyethylene glycol in the printing material, resulting in a porous and flexible β-TCP / 90:10 caprolactone / glycolic acid copolymer composite. The scaffold was dried for at least 12 hours to ensure that any residual water had evaporated from the porous scaffold before binding with a therapeutic agent (such as tBMP2). The scaffold was sterilized by soaking in a 70% ethanol solution for 2–4 hours and then dried in a biosafety cabinet for approximately 12 hours. Images of the scaffold are shown below. Figures 3A-3C .

[0462] 3D printing was also performed using an FFF 3D printer. Ink #3 was extruded into a filament, which was then loaded into the FFF 3D printer to create a 3D-printed scaffold. The scaffold was then processed using the post-processing methods described above.

[0463] Ink formulations and scaffolds #4

[0464] Method: To prepare 2.5cc batches of ink, 2.8 g of β-TCP powder, 0.94 g of 50:50 poly(D,L-lactide-co-glycolic acid) copolymer blocks, and 0.94 g of polyethylene glycol flakes were added to a glass mixing container. The wide-mouthed glass flask was placed in a FlackTek speedmixer and mixed at low intensity (300 rpm) for 2 min to homogenize the powder blend before high-speed mixing. It was then mixed at high intensity (3500 rpm) for 5 min. During mixing, internal friction caused the 50:50 poly(D,L-lactide-co-glycolic acid) copolymer and polyethylene glycol to flow, turning the ink into a viscous molten liquid. This liquid-phase mixing promoted the tight dispersion of the β-TCP powder into the molten polymer blend. The blended ink was allowed to cool for 10–15 min and then mixed again at 3500 rpm for 5 min. Repeat the mixing / cooling process a total of four times, mixing for 5 minutes at 3500 rpm. After the fourth mixing at 3500 rpm, pour the ink feed onto a glass plate and use two scrapers to form a cylinder approximately 1 cm in diameter × 3 cm in length. While the ink is still semi-molten, cut it into several slices approximately 1-2 cm long using a straight razor.

[0465] 3D Printing: Transfer the solid polymer / β-TCP sheet into a 5cc stainless steel syringe (for an Allevi 3 bioprinter). Heat the extruder CORE printhead to 85°C and hold for approximately 30 minutes to ensure the ink melts. Print the ink using a 400-micron inner diameter conical metal Luer head, applying 60 psi pressure and a nozzle speed of 7 mm / s. 3D print the scaffold onto a piece of craft tape applied to a smooth glass or polymer surface (such as a glass microscope slide, a large glass plate, or a 96-well plate cover).

[0466] Post-processing: The 3D-printed structure was soaked overnight in distilled water to dissolve the polyethylene glycol in the printing material, resulting in a porous and flexible β-TCP / 50:50 poly(D,L-lactide-co-glycolic acid) composite. The scaffold was dried for at least 12 hours to ensure that any residual water had evaporated from the porous scaffold before binding with a therapeutic agent (such as tBMP2). The scaffold was sterilized by soaking in a 70% ethanol solution for 2–4 hours and then dried in a biosafety cabinet for approximately 12 hours. Images of the scaffold are shown below. Figures 4A-4C .

[0467] 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 create a 3D-printed scaffold. The scaffold was then processed using the post-processing methods described above.

[0468] Ink formulations and scaffolds #5

[0469] Method: To prepare 5cc batches of ink, 5.6g of β-TCP powder, 1.87g of polycaprolactone powder, and 1.87g of polyethylene glycol flakes were added to a glass mixing container. The wide-mouth glass flask was placed in a double asymmetric centrifugal mixer (FlackTek Speedmixer) and mixed at low intensity (300 rpm) for 2 min to homogenize the powder blend before mixing at high rpm. It was then mixed at high intensity (3500 rpm) for 5 min. During mixing, internal friction caused the polycaprolactone and polyethylene glycol to melt, turning the ink into a viscous molten liquid. This liquid-phase mixing promoted the tight dispersion of the β-TCP powder into the molten polymer blend. The molten ink was poured onto a glass plate and flattened into a layer of approximately 3mm thickness using a doctor blade for cooling. After cooling, it was cut into granules of approximately 3-4mm using a shearing machine. This process was repeated for two additional 5cc batches to produce a total of 15cc granules for filament extrusion.

[0470] Filament manufacturing: Load 15cc of 3-4mm granules into the hopper of the filament extruder (Filabot EX2 filament extruder). Use a melt filter nozzle with 3 x length extensions (remove the filter screen) and a 1.75mm diameter orifice. Place a cooling fan near the extrusion nozzle to accelerate the curing of the extruded filament. Extrude the 1.75mm diameter filament at 1 / 2 speed on the simulated dial (approximately 1cm / s) at an extrusion temperature of 62°C.

[0471] 3D Printing: A 1.75mm 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 15mm / s.

[0472] Post-processing: The 3D-printed structure was soaked overnight in distilled water to dissolve the polyethylene glycol in the printing material, resulting in a porous and flexible β-TCP / polycaprolactone composite. The scaffold was dried for at least 12 hours to ensure that any residual water had evaporated from the porous scaffold before binding with the therapeutic agent (such as tBMP2). The scaffold was sterilized by soaking in a 70% ethanol solution for 2–4 hours and then dried in a biosafety cabinet for approximately 12 hours. Images of the scaffold are shown below. Figures 5A-5C .

[0473] Ink formulations and scaffolds #6

[0474] Methods: To prepare 5.5cc batches of ink, 5.6g of β-TCP powder, 1.87g of polycaprolactone powder, 1.87g of polyethylene glycol flakes, and 1.04g of sodium bicarbonate were added to a glass mixing container. The wide-mouthed glass flask was placed in a FlackTek speedmixer and mixed at low intensity (300 rpm) for 2 min to homogenize the powder blend before mixing at high rpm. It was then mixed at high intensity (3500 rpm) for 2 min. During mixing, internal friction caused the polycaprolactone and polyethylene glycol to melt, turning the ink into a viscous molten liquid. This liquid-phase mixing promoted the tight dispersion of the β-TCP powder and sodium bicarbonate powder into the molten polymer blend. The molten ink was poured onto a glass plate and flattened with a scraper to a layer approximately 3mm thick for cooling. After cooling, it was cut into granules of approximately 3-4mm using a shearing machine. Repeat this process for two additional 5.5cc batches to produce a total of 16.5cc of pellets for filament extrusion.

[0475] Filament Manufacturing: Load 16.5cc of 3-4mm granules into the hopper of the filament extruder (Filabot EX2 filament extruder). Use a melt filter nozzle with 3x length extensions (remove the filter screen) and a 1.75mm diameter orifice. Place a cooling fan near the extrusion nozzle to accelerate the curing of the extruded filament. Extrude the 1.75mm diameter filament at 1 / 2 speed on the simulated dial (approximately 1cm / s) at an extrusion temperature of 62°C.

[0476] 3D Printing: A 1.75mm 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 15mm / s.

[0477] Post-processing: The 3D-printed structure was soaked overnight in distilled water to dissolve polyethylene glycol and sodium carbonate (a byproduct of the thermal decomposition of sodium bicarbonate) in the foam-like printing material, resulting in a porous and flexible β-TCP / polycaprolactone composite. The scaffold was dried for at least 12 hours to ensure that any residual water had evaporated from the porous scaffold before binding with the therapeutic agent (such as tBMP2). The scaffold was sterilized by soaking in a 70% ethanol solution for 2–4 hours and then dried in a biosafety cabinet for approximately 12 hours. Images of the scaffold are shown below. Figures 6A-6C .

[0478] Ink formulations and scaffolds #7

[0479] Methods: To prepare 10cc batches of ink, 11.2 g of β-TCP powder, 3.74 g of p-dioxanone / L-lactide (90:10) copolymer fragments, and 3.74 g of polyethylene glycol flakes were added to a glass mixing container. The glass flask was placed in a FlackTek speedmixer and mixed at low intensity (300 rpm) for 2 min to homogenize the powder blend before high-speed mixing. The flask was then transferred to a hot plate and heated until it reached 185°C (measured with an IR thermometer). Immediately afterwards, the flask was transferred back to the speedmixer and mixed at high intensity (3500 rpm) for 5 min. Liquid-phase mixing promoted the tight dispersion of the β-TCP powder into the molten polymer blend. The flask was then transferred back to the hot plate, and the temperature was increased to 185°C. Upon reaching this temperature, immediately transfer the wide-mouthed glass bottle back to the double asymmetric centrifugal mixer and mix for another 5 minutes at 3500 rpm. Repeat the mixing / heating process for a total of four 5-minute mixtures at 3500 rpm. After the fourth mixture at 3500 rpm, pour the ink feed onto a glass plate and use two spatulas to form a cylinder approximately 1 cm in diameter × 6 cm in length. While the ink is still semi-molten, cut it into several slices approximately 1-2 cm long using a straight razor.

[0480] 3D Printing: Transfer the solid polymer / β-TCP sheet into a 5cc stainless steel syringe (for an Allevi 3 bioprinter). Heat the extruder CORE printhead to 110°C and hold for approximately 30 minutes to ensure the ink melts. Print the ink using a 400-micron inner diameter conical metal Luer head, applying 15 psi pressure and a nozzle speed of 10 mm / s. 3D print the scaffold onto a piece of craft tape applied to a smooth glass or polymer surface (such as a glass microscope slide, a large glass plate, or a 96-well plate cover).

[0481] Post-processing: The 3D-printed structure was soaked overnight in distilled water to dissolve the polyethylene glycol in the printing material. This process produces a porous and flexible β-TCP / 90:10 dioxane-L-lactide copolymer composite. The scaffold was dried for at least 12 hours to ensure that any residual water had evaporated from the porous scaffold before binding with therapeutic agents such as tBMP2 protein. The scaffold was sterilized by soaking in a 70% ethanol solution for 2–4 hours and then dried in a biosafety cabinet for approximately 12 hours. Images of the scaffold are shown below. Figures 7A-7C .

[0482] 3D printing was also performed using an FFF 3D printer. Ink #7 was extruded into a filament, which was then loaded into the FFF 3D printer to create a 3D-printed scaffold. The scaffold was then processed using the post-processing methods described above.

[0483] Ink formulations and scaffolds #8

[0484] Methods: To prepare 16cc batches of ink, 18g of β-TCP powder, 6g of polycaprolactone powder, 3g of polyethylene glycol (8,000MW) flakes, and 3g of polyethylene glycol (20,000MW) flakes were added to a Teflon mixing container. The Teflon container was placed in a FlackTek speedmixer and mixed at low intensity (300rpm) for 2 min to homogenize the powder blend before mixing at high rpm. Then, the Teflon container was mixed at high intensity (3500rpm) for 2.5 min. During mixing, internal friction caused the polycaprolactone and polyethylene glycol to melt, turning the ink into a viscous molten liquid. This liquid-phase mixing promoted the tight dispersion of the β-TCP powder into the molten polymer blend. The molten ink was poured onto a glass plate and flattened into a layer approximately 3mm thick using a doctor blade for cooling. It was cooled for 10–15 min. Return the mixture to the Teflon container and mix again at high intensity (3500 rpm) for 2.5 minutes. Repeat the mixing / cooling process for a total of four 2.5-minute mixtures at 3500 rpm. After cooling the ink following the fourth and final mixtures, cut it into pellets of approximately 3-4 mm using a shearing machine.

[0485] Filament Manufacturing: Load 16cc of 3-4mm granules into the hopper of the filament extruder (Filabot EX2 filament extruder). Use a melt filter nozzle with 3x length extensions (remove the filter screen) and a 1.75mm diameter orifice. Place a cooling fan near the extrusion nozzle to accelerate the curing of the extruded filament. Extrude the 1.75mm diameter filament at 1 / 2 speed on the simulated dial (approximately 1cm / s) at an extrusion temperature of 62°C.

[0486] 3D Printing: A 1.75mm 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 15mm / s.

[0487] Post-processing: The 3D-printed structure was soaked overnight in distilled water to dissolve the polyethylene glycol in the printing material, resulting in a porous and flexible β-TCP / polycaprolactone composite. The scaffold was dried for at least 12 hours to ensure that any residual water had evaporated from the porous scaffold before binding with the therapeutic agent (such as tBMP2). The scaffold was sterilized by soaking in a 70% ethanol solution for 2–4 hours and then dried in a biosafety cabinet for approximately 12 hours. Images of the scaffold are shown below. Figures 8A-8C .

[0488] Ink formulations and scaffolds #9

[0489] Methods: To prepare 16.3 cc batches 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 container. The Teflon container was placed in a FlackTek speedmixer and mixed at low intensity (300 rpm) for 2 min to homogenize the powder blend before mixing at high rpm. The Teflon container was then mixed at high intensity (3500 rpm) for 2 min. During mixing, internal friction caused the polycaprolactone and polyethylene glycol to melt, turning the ink into a viscous molten liquid. This liquid-phase mixing promoted the tight dispersion of the β-TCP powder into the molten polymer blend. The molten ink was poured onto a glass plate and flattened into a layer approximately 3 mm thick using a doctor blade for cooling. Cool it for 10-15 minutes. Return the mixture to the Teflon container and mix again at high intensity (3500 rpm) for 2 minutes. Repeat the mixing / cooling process for a total of four 2-minute mixtures at 3500 rpm. After cooling the ink following the fourth and final mixtures, cut it into pellets of approximately 3-4 mm using a shear.

[0490] Filament Manufacturing: Load 16.3cc of 3-4mm granules into the hopper of the filament extruder (Filabot EX2 filament extruder). Use a melt filter nozzle with 3x length extensions (remove the filter screen) and a 1.75mm diameter orifice. Place a cooling fan near the extrusion nozzle to accelerate the curing of the extruded filament. Extrude the 1.75mm diameter filament at 1 / 2 speed on the simulated dial (approximately 1cm / s) at an extrusion temperature of 62°C.

[0491] 3D Printing: A 1.75mm 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 10mm / s.

[0492] Post-processing: The 3D-printed structure was soaked overnight in distilled water to dissolve the polyethylene glycol and sucrose in the printing material, resulting in a porous and flexible β-TCP / polycaprolactone composite. The scaffold was dried for at least 12 hours to ensure that any residual water had evaporated from the porous scaffold before binding with the therapeutic agent (such as tBMP2). The scaffold was sterilized by soaking in a 70% ethanol solution for 2–4 hours and then dried in a biosafety cabinet for approximately 12 hours. Images of the scaffold are shown below. Figures 9A-9C .

[0493] Ink formulations and scaffolds #10

[0494] Methods: To prepare 16cc batches of ink, 18g of β-TCP powder, 6g of caprolactone / glycolic acid copolymer (95:5) granules, 3g of polyethylene glycol (8,000MW) flakes, and 3g of polyethylene glycol (20,000MW) flakes were added to a Teflon mixing container. The Teflon container was placed in a FlackTek speedmixer and mixed at low intensity (300rpm) for 2 min to homogenize the powder blend before high-speed mixing. The Teflon container was then mixed at high intensity (3500rpm) for 2.5 min. During mixing, internal friction caused the caprolactone / glycolic acid copolymer (95:5) and polyethylene glycol to melt, turning the ink into a viscous molten liquid. This liquid-phase mixing promoted the tight dispersion of the β-TCP powder into the molten polymer blend. The molten ink was poured onto a glass plate and flattened into a layer approximately 3mm thick using a doctor blade for cooling. Cool it for 10-15 minutes. Return the mixture to the Teflon container and mix again at high intensity (3500 rpm) for 4 minutes. Repeat the mixing / cooling process, for a total of one 2.5-minute mix at 3500 rpm and three 4-minute mixes at 3500 rpm. After cooling the ink following the fourth and final mixes, cut it into pellets of approximately 3-4 mm using a shear.

[0495] Filament Manufacturing: Load 16cc of 3-4mm granules into the hopper of the filament extruder (Filabot EX2 filament extruder). Use a melt filter nozzle with 3x length extensions (remove the filter screen) and a 1.75mm diameter orifice. Place a cooling fan near the extrusion nozzle to accelerate the curing of the extruded filament. Extrude the 1.75mm diameter filament at 1 / 2 speed on the simulated dial (approximately 1cm / s) at an extrusion temperature of 62°C.

[0496] 3D Printing: A 1.75mm 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 10mm / s.

[0497] Post-processing: The 3D-printed structure was soaked overnight in distilled water to dissolve the polyethylene glycol in the printing material, resulting in a porous and flexible β-TCP / caprolactone / glycolic acid copolymer (95:5) composite. The scaffold was dried for at least 12 hours to ensure that any residual water had evaporated from the porous scaffold before binding with a therapeutic agent (such as tBMP2). The scaffold was sterilized by soaking in a 70% ethanol solution for 2–4 hours and then dried in a biosafety cabinet for approximately 12 hours. Images of the scaffold are shown below. Figures 10A-10C .

[0498] Ink formulations and scaffolds #11

[0499] Methods: To prepare 16cc batches of ink, 18g of β-TCP powder, 6g of caprolactone / glycolic acid copolymer (90:10) fragments, 3g of polyethylene glycol (8,000MW) flakes, and 3g of polyethylene glycol (20,000MW) flakes were added to a Teflon mixing container. The Teflon container was placed in a FlackTek speedmixer and mixed at low intensity (300rpm) for 2 min to homogenize the powder blend before high-speed mixing. The Teflon container was then mixed at high intensity (3500rpm) for 2 min. During mixing, internal friction caused the caprolactone / glycolic acid copolymer (90:10) and polyethylene glycol to melt, turning the ink into a viscous molten liquid. This liquid-phase mixing promoted the tight dispersion of the β-TCP powder into the molten polymer blend. The molten ink was poured onto a glass plate and flattened into a layer approximately 3mm thick using a doctor blade for cooling. Cool it for 10-15 minutes. Return the mixture to the Teflon container and mix again at high intensity (3500 rpm) for 5 minutes. Repeat the mixing / cooling process, for a total of one 2-minute mix at 3500 rpm and three 5-minute mixes at 3500 rpm. After cooling the ink after the fourth and final mixes, cut it into pellets of approximately 3-4 mm using a shear.

[0500] Filament Manufacturing: Load 16cc of 3-4mm granules into the hopper of the filament extruder (Filabot EX2 filament extruder). Use a melt filter nozzle with 3x length extensions (remove the filter screen) and a 1.75mm diameter orifice. Place a cooling fan near the extrusion nozzle to accelerate the curing of the extruded filament. Extrude the 1.75mm diameter filament at 1 / 2 speed on the simulated dial (approximately 1cm / s) at an extrusion temperature of 62°C.

[0501] 3D Printing: A 1.75mm 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 10mm / s.

[0502] Post-processing: The 3D-printed structure was soaked overnight in distilled water to dissolve the polyethylene glycol in the printing material, resulting in a porous and flexible β-TCP / caprolactone / glycolic acid copolymer (90:10) composite. The scaffold was dried for at least 12 hours to ensure that any residual water had evaporated from the porous scaffold before binding with a therapeutic agent (such as tBMP2). The scaffold was sterilized by soaking in a 70% ethanol solution for 2–4 hours and then dried in a biosafety cabinet for approximately 12 hours. Images of the scaffold are shown below. Figures 11A-11C .

[0503] Ink formulations and scaffolds #12

[0504] Methods: To prepare 8cc batches of ink, 9g of β-TCP powder, 3g of poly(D,L-lactide-co-glycolic acid) copolymer (50:50) blocks, 1.5g of polyethylene glycol (8,000MW) flakes, and 1.5g of polyethylene glycol (20,000MW) flakes were added to a Teflon mixing container. The Teflon container was placed in a FlackTek Speedmixer and mixed at low intensity (300rpm) for 2 min to homogenize the powder blend before mixing at high rpm. The Teflon container was then mixed at high intensity (3500rpm) for 2 min. During mixing, internal friction caused the poly(D,L-lactide-co-glycolic acid) copolymer (50:50) and polyethylene glycol to melt, turning the ink into a viscous molten liquid. This liquid-phase mixing promoted the tight dispersion of the β-TCP powder into the molten polymer blend. Pour the molten ink onto a glass plate and flatten it into a layer approximately 3 mm thick using a scraper for cooling. Cool it for 10-15 minutes. Return the mixture to the Teflon container and mix again at high intensity (3500 rpm) for 2.5 minutes. Repeat the mixing / cooling process, for a total of one 2-minute mix at 3500 rpm and two 2.5-minute mixes at 3500 rpm. After cooling the ink following the third and final mixes, cut it into granules approximately 3-4 mm using a shear.

[0505] Filament Manufacturing: Load 8cc of 3-4mm granules into the hopper of the filament extruder (Filabot EX2 filament extruder). Use a melt filter nozzle with 3x length extensions (remove the filter screen) and a 1.75mm diameter orifice. Place a cooling fan near the extrusion nozzle to accelerate the curing of the extruded filament. Extrude the 1.75mm diameter filament at 1 / 2 speed on the simulated dial (approximately 1cm / s) at an extrusion temperature of 62°C.

[0506] 3D Printing: A 1.75mm 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 10mm / s.

[0507] Post-processing: The 3D-printed structure was soaked overnight in distilled water to dissolve the polyethylene glycol in the printing material, resulting in a porous and flexible β-TCP / poly(D,L-lactide-co-glycolic acid) copolymer (50:50) composite. The scaffold was dried for at least 12 hours to ensure that any residual water had evaporated from the porous scaffold before binding with a therapeutic agent (such as tBMP2). The scaffold was sterilized by soaking in a 70% ethanol solution for 2–4 hours and then dried in a biosafety cabinet for approximately 12 hours. Images of the scaffold are shown below. Figures 12A-12C .

[0508] Ink formulations and scaffolds #13

[0509] Ink #13 is printed using a syringe-based melt extrusion printing method, for example, using the method described for printing inks #1-#4. Ink #13 is also printed using filament fabrication 3D printing, for example, using the method described for inks #5 and #6.

[0510] Ink formulations and scaffolds #14

[0511] Ink #14 is printed using a syringe-based melt extrusion printing method, for example, using the method described for printing inks #1-#4. Ink #14 is also printed using filament fabrication 3D printing, for example, using the method described for inks #5 and #6.

[0512] Ink formulations and scaffolds #15

[0513] Ink #9 is printed using a syringe-based melt extrusion printing method, for example, using the method described for printing inks #1-#4. Ink #15 is printed using filament fabrication 3D printing, for example, using the method described for inks #5 and #6.

[0514] Structural characteristics

[0515] The physical properties of stents #1-#6 were determined and summarized in Table 19.

[0516] Table 19. Characteristics of the Exemplary Support

[0517] Ink #1 1.45 30 434 Ink #2 1.56 23 397 Ink #3 1.70 25 569 Ink #4 1.49 32 464 Ink #5 1.23 39 378 Ink #6 1.22 39 392 Ink #7 1.50 33 499 Ink #8 1.22 37 359 Ink #9 1.23 37 351 Ink #10 1.28 37 374 Ink #11 1.45 29 335 Ink #12 1.30 37 364

[0518] The structure of this embodiment was tested using Brunauer-Emmett-Teller (BET) surface area analysis via gas physical adsorption.

[0519] Compression tests were also conducted on the structure.

[0520] Example 3: Therapeutic agent

[0521] A chimeric polypeptide comprising five β-tricalcium phosphate-binding peptides linked to therapeutic BMP peptides was expressed and purified using standard expression and purification methods. This chimeric polypeptide, designated tBMP-2, has the following sequence: MPIGSLLADTTHHRPWTVIGESTHHRPWSIIGESSHHKPFTGLGDTTHHRPWGILAESTHHKPWTASGAGGSEGGGSEGGTSGATGAGTSTSGGGASTGGGTGQAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR (SEQ ID NO:434).

[0522] Example 4: Device Manufacturing

[0523] A 3D-printed structure was combined with tBMP-2 therapeutic agent to create a device. A 0.75 mg / mL tBMP-2 binding solution (10 mM sodium acetate, 7 mM acetic acid, 100 mM NaCl, pH 4.75) was prepared and sterilized using a 0.22 μm filter. In a biosafety cabinet, the tBMP-2 binding solution was added to sterile polypropylene tubes using sterile pipettes to achieve a ratio of 15 mg tBMP-2 per g of 3D-printed scaffold. The sterile scaffold was added to the binding solution using sterile forceps, and the polypropylene tube was then sealed and wrapped with paraffin film. The tube was placed on a LabLine Instruments titration plate shaker at speed 6 for 2 hours. In the biosafety cabinet, the scaffold + tBMP-2 was removed using sterile forceps and placed in a separate sterile polypropylene tube containing sterile PBS (the same volume as the tBMP-2 binding solution). The tube was capped, wrapped with paraffin film, and returned to the titration plate shaker at speed 6 for 5 minutes. In a biosafety cabinet, open the tube, remove the scaffold + tBMP-2 using sterile forceps, and place it in a sterile Petri dish. Allow the scaffold + tBMP-2 to dry overnight in the biosafety cabinet to obtain the tBMP-2 device.

[0524] The mass of tBMP-2 remaining in the binding solution and the mass of tBMP-2 in the PBS washing solution were measured using an A280 absorbance meter. The sum of these masses was calculated and then subtracted from the initial mass of tBMP-2 in the binding solution to obtain the mass of tBMP-2 retained on the 3D-printed scaffold.

[0525] Example 5: Animal Model

[0526] The practicality of the embodiments described herein is demonstrated by showcasing bone regeneration in animal models (e.g., rats, rabbits, goats, pigs, dogs, sheep) and evaluated by μCT imaging and histological analysis. Indications may include lumbar fusion (3D-printed inserts for spinal fusion devices), posterolateral (PLF) spinal fusion (3D-printed scaffolds across transverse processes), tibial segmental defects (3D-printed scaffolds based on patient CT data), and / or alveolar ridge elevation (3D-printed thin barrier membranes). sequence list <110> Treatment and Adjustment Company <120> Ceramic Compositions and Application Methods <130> 50222-709.601 <140> <141> <160> 501 <170> PatentIn version 3.5 <210> 1 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 1 Leu Leu Ala Asp Thr Thr His His Arg Pro Trp Thr 1 5 10 <210> 2 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 2 Val Ile Gly Glu Ser Thr His His Arg Pro Trp Ser 1 5 10 <210> 3 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 3 Leu Ile Ala Asp Ser Thr His His Ser Pro Trp Thr 1 5 10 <210> 4 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 4 Ile Leu Ala Glu Ser Thr His His Lys Pro Trp Thr 1 5 10 <210> 5 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 5 Ile Leu Ala Glu Thr Thr His His Arg Pro Trp Ser 1 5 10 <210> 6 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 6 Ile Ile Gly Glu Ser Ser His His Lys Pro Phe Thr 1 5 10 <210> 7 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 7 Gly Leu Gly Asp Thr Thr His His Arg Pro Trp Gly 1 5 10 <210> 8 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 8 Val Leu Gly Asp Thr Thr His His Lys Pro Trp Thr 1 5 10 <210> 9 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 9 Ile Val Ala Asp Ser Thr His His Arg Pro Trp Thr 1 5 10 <210> 10 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 10 Ser Thr Ala Asp Thr Ser His His Arg Pro Ser 1 5 10 <210> 11 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 11 Thr Ser Gly Gly Glu Ser Thr His His Arg Pro Ser 1 5 10 <210> 12 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 12 Thr Ser Gly Gly Glu Ser Ser His His Lys Pro Ser 1 5 10 <210> 13 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 13 Thr Gly Ser Gly Asp Ser Ser His His Arg Pro Ser 1 5 10 <210> 14 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 14 Gly Ser Ser Gly Glu Ser Thr His His Lys Pro Ser Thr 1 5 10 <210> 15 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 15 Val Gly Ala Asp Ser Thr His His Arg Pro Val Thr 1 5 10 <210> 16 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 16 Gly Ala Ala Asp Thr Thr His His Arg Pro Val Thr 1 5 10 <210> 17 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 17 Ala Gly Ala Asp Thr Thr His His Arg Pro Val Thr 1 5 10 <210> 18 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 18 Gly Gly Ala Asp Thr Thr His His Arg Pro Ala Thr 1 5 10 <210> 19 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 19 Gly Gly Ala Asp Thr Thr His His Arg Pro Gly Thr 1 5 10 <210> 20 <211> twenty four <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 20 Leu Leu Ala Asp Thr Thr His His Arg Pro Trp Thr Val Ile Gly Glu 1 5 10 15 Ser Thr His His Arg Pro Trp Ser 20 <210> twenty one <211> 36 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic polypeptide <400> twenty one Leu Leu Ala Asp Thr Thr His His Arg Pro Trp Thr Val Ile Gly Glu 1 5 10 15 Ser Thr His His Arg Pro Trp Ser Ile Ile Gly Glu Ser Ser His His 20 25 30 Lys Pro Phe Thr 35 <210> twenty two <211> 60 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic polypeptide <400> twenty two Leu Leu Ala Asp Thr Thr His His Arg Pro Trp Thr Val Ile Gly Glu 1 5 10 15 Ser Thr His His Arg Pro Trp Ser Ile Ile Gly Glu Ser Ser His His 20 25 30 Lys Pro Phe Thr Gly Leu Gly Asp Thr Thr His His Arg Pro Trp Gly 35 40 45 Ile Leu Ala Glu Ser Thr His His Lys Pro Trp Thr 50 55 60 <210> twenty three <211> twenty four <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> twenty three Leu Leu Ala Asp Thr Thr His His Arg Pro Trp Thr Ile Leu Ala Glu 1 5 10 15 Ser Thr His His Lys Pro Trp Thr 20 <210> twenty four <211> 60 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic polypeptide <400> twenty four Leu Leu Ala Asp Thr Thr His His Arg Pro Trp Thr Ile Leu Ala Glu 1 5 10 15 Ser Thr His His Lys Pro Trp Thr Leu Leu Ala Asp Thr Thr His His 20 25 30 Arg Pro Trp Thr Ile Leu Ala Glu Ser Thr His His Lys Pro Trp Thr 35 40 45 Leu Leu Ala Asp Thr Thr His His Arg Pro Trp Thr 50 55 60 <210> 25 <211> twenty four <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 25 Leu Leu Ala Asp Thr Thr His His Arg Pro Trp Thr Gly Leu Gly Asp 1 5 10 15 Thr Thr His His Arg Pro Trp Gly 20 <210> 26 <211> 36 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic polypeptide <400> 26 Leu Leu Ala Asp Thr Thr His His Arg Pro Trp Thr Gly Leu Gly Asp 1 5 10 15 Thr Thr His His Arg Pro Trp Gly Leu Leu Ala Asp Thr Thr His His 20 25 30 Arg Pro Trp Thr 35 <210> 27 <211> 60 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic polypeptide <400> 27 Leu Leu Ala Asp Thr Thr His His Arg Pro Trp Thr Gly Leu Gly Asp 1 5 10 15 Thr Thr His His Arg Pro Trp Gly Leu Leu Ala Asp Thr Thr His His 20 25 30 Arg Pro Trp Thr Gly Leu Gly Asp Thr Thr His His Arg Pro Trp Gly 35 40 45 Leu Leu Ala Asp Thr Thr His His Arg Pro Trp Thr 50 55 60 <210> 28 <211> 84 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic polypeptide <400> 28 Leu Leu Ala Asp Thr Thr His His Arg Pro Trp Thr Gly Leu Gly Asp 1 5 10 15 Thr Thr His His Arg Pro Trp Gly Leu Leu Ala Asp Thr Thr His His 20 25 30 Arg Pro Trp Thr Gly Leu Gly Asp Thr Thr His His Arg Pro Trp Gly 35 40 45 Leu Leu Ala Asp Thr Thr His His Arg Pro Trp Thr Gly Leu Gly Asp 50 55 60 Thr Thr His His Arg Pro Trp Gly Leu Leu Ala Asp Thr Thr His His 65 70 75 80 Arg Pro Trp Thr <210> 29 <211> 60 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic polypeptide <400> 29 Ser Thr Ala Asp Thr Ser His His Arg Pro Ser Thr Ser Gly Gly Glu 1 5 10 15 Ser Thr His His Arg Pro Ser Thr Ser Gly Gly Glu Ser Ser His His 20 25 30 Lys Pro Ser Thr Gly Ser Gly Asp Ser Ser His His Arg Pro Ser Gly 35 40 45 Ser Ser Gly Glu Ser Thr His His Lys Pro Ser Thr 50 55 60 <210> 30 <211> 60 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic Polypeptide <400> 30 Val Gly Ala Asp Ser Thr His His Arg Pro Val Thr Gly Ala Ala Asp 1 5 10 15 Thr Thr His His Arg Pro Val Thr Ala Gly Ala Asp Thr Thr His His 20 25 30 Arg Pro Val Thr Gly Gly Ala Asp Thr Thr His His Arg Pro Ala Thr 35 40 45 Gly Gly Ala Asp Thr Thr His His Arg Pro Gly Thr 50 55 60 <210> 31 <211> 108 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: Synthetic Polypeptide <400> 31 Ser Thr Ala Asp Thr Ser His His Arg Pro Ser Leu Leu Ala Asp Thr 1 5 10 15 Thr His His Arg Pro Trp Thr Thr Ser Gly Gly Glu Ser Thr His His 20 25 30 Arg Pro Ser Val Gly Ala Asp Ser Thr His His Arg Pro Val Thr Thr 35 40 45 Ser Gly Gly Glu Ser Ser His His Lys Pro Ser Gly Ala Ala Asp Thr 50 55 60 Thr His His Arg Pro Val Thr Thr Gly Ser Gly Asp Ser Ser His His 65 70 75 80 Arg Pro Ser Gly Ser Ser Ser Gly Glu Ser Thr His His Lys Pro Ser Thr 85 90 95 Gly Gly Ala Asp Thr Thr His His Arg Pro Ala Thr 100 105 <210> 32 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 32 Ala Ala Ala Asp Thr Thr His His Arg Pro Trp Thr 1 5 10 <210> 33 <211> 60 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic polypeptide <400> 33 Ala Ala Ala Asp Thr Thr His His Arg Pro Trp Thr Ala Ala Ala Asp 1 5 10 15 Thr Thr His His Arg Pro Trp Thr Ala Ala Ala Ala Asp Thr Thr His His 20 25 30 Arg Pro Trp Thr Ala Ala Ala Asp Thr Thr His His Arg Pro Trp Thr 35 40 45 Ala Ala Ala Asp Thr Thr His His Arg Pro Trp Thr 50 55 60 <210> 34 <211> 60 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic polypeptide <400> 34 Leu Leu Ala Asp Ala Ala His His Arg Pro Trp Thr Leu Leu Ala Asp 1 5 10 15 Ala Ala His His Arg Pro Trp Thr Leu Leu Ala Asp Ala Ala His His 20 25 30 Arg Pro Trp Thr Leu Leu Ala Asp Ala Ala His His Arg Pro Trp Thr 35 40 45 Leu Leu Ala Asp Ala Ala His His Arg Pro Trp Thr 50 55 60 <210> 35 <211> 60 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic polypeptide <400> 35 Leu Leu Ala Asp Thr Thr Ala Ala Arg Pro Trp Thr Leu Leu Ala Asp 1 5 10 15 Thr Thr Ala Ala Arg Pro Trp Thr Leu Leu Ala Asp Thr Thr Ala Ala 20 25 30 Arg Pro Trp Thr Leu Leu Ala Asp Thr Thr Ala Ala Arg Pro Trp Thr 35 40 45 Leu Leu Ala Asp Thr Thr Ala Ala Arg Pro Trp Thr 50 55 60 <210> 36 <211> twenty four <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 36 Leu Leu Ala Asp Thr Thr His His Arg Pro Trp Thr Leu Leu Ala Asp 1 5 10 15 Thr Thr His His Arg Pro Trp Thr 20 <210> 37 <211> 36 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic polypeptide <400> 37 Leu Leu Ala Asp Thr Thr His His Arg Pro Trp Thr Leu Leu Ala Asp 1 5 10 15 Thr Thr His His Arg Pro Trp Thr Leu Leu Ala Asp Thr Thr His His 20 25 30 Arg Pro Trp Thr 35 <210> 38 <211> 60 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic polypeptide <400> 38 Leu Leu Ala Asp Thr Thr His His Arg Pro Trp Thr Leu Leu Ala Asp 1 5 10 15 Thr Thr His His Arg Pro Trp Thr Leu Leu Ala Asp Thr Thr His His 20 25 30 Arg Pro Trp Thr Leu Leu Ala Asp Thr Thr His His Arg Pro Trp Thr 35 40 45 Leu Leu Ala Asp Thr Thr His His Arg Pro Trp Thr 50 55 60 <210> 39 <211> 120 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic polypeptide <400> 39 Ser Thr Ser Gly Ser Thr Val Ile Gly Glu Ser Thr His His Arg Pro 1 5 10 15 Trp Ser Leu Ile Ala Asp Ser Thr His His Ser Pro Trp Thr Ile Leu 20 25 30 Ala Glu Ser Thr His His Lys Pro Trp Thr Ile Leu Ala Glu Thr Thr 35 40 45 His His Arg Pro Trp Ser Ile Ile Gly Glu Ser Ser His His Lys Pro 50 55 60 Phe Thr Gly Leu Gly Asp Thr Thr His His Arg Pro Trp Gly Val Leu 65 70 75 80 Gly Asp Thr Thr His His Lys Pro Trp Thr Ile Val Ala Asp Ser Thr 85 90 95 His His Arg Pro Trp Thr Gly Gln Val Leu Pro Thr Thr Thr Pro Ser 100 105 110 Ser Pro Ser Thr Thr Ser Gly Ser 115 120 <210> 40 <211> 48 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic polypeptide <400> 40 Leu Leu Ala Asp Thr Thr His His Arg Pro Trp Thr Val Ile Gly Glu 1 5 10 15 Ser Thr His His Arg Pro Trp Ser Ile Ile Gly Glu Ser Ser His His 20 25 30 Lys Pro Phe Thr Gly Leu Gly Asp Thr Thr His His Arg Pro Trp Gly 35 40 45 <210> 41 <211> 48 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic polypeptide <400> 41 Val Ile Gly Glu Ser Thr His His Arg Pro Trp Ser Ile Ile Gly Glu 1 5 10 15 Ser Ser His His Lys Pro Phe Thr Gly Leu Gly Asp Thr Thr His His 20 25 30 Arg Pro Trp Gly Ile Leu Ala Glu Ser Thr His His Lys Pro Trp Thr 35 40 45 <210> 42 <211> 132 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic polypeptide <220> <221> MOD_RES <222> (13)..(132) <223> This region may contain the following sequences: One: "LLADTTHHRPWT" or "VIGESTHHRPWS" or "LIADSTHHSPWT" Or "ILAESTHHKPWT" or "ILAETTHHRPWS" or "IIGESSHHKPFT" or "GLGDTTHHRPWG" or "VLGDTTHHKPWT" or "IVADSTHHRPWT" or <220> <221> MOD_RES <222> (13)..(132) <223> Continued from above: "STADTSHHRPS" or "TSGGESTHHRPS" or "TSGGESSHHKPS" or "TGSGDSSHHRPS" or "GSSGESTHHKPST" or "VGADSTHHRPVT" or "GAADTTHHRPVT" or "AGADTTHHRPVT" or "GGADTTHHRPAT" or "GGADTTHHRPGT" or <220> <221> MOD_RES <222> (13)..(132) <223> Continuing from above: "LLADTTHHRPWTVIGESTHHRPWS" or "LLADTTHHRPWTVIGESTHHRPWSIIGESSHHKPFT" or "LLADTTHHRPWTVIGESTHHRPWSIIGESSHHKPFTGLGDTTHRPWGILAESTHHK PWT or "LLADTTHHRPWTILAESTHHKPWT" or "LLADTTHHRPWTILAESTHHK" <220> <221> MOD_RES <222> (13)..(132) <223> Continued from above: PWTLLADTTHHRPWTILAESTHHKPWTLLADTTHHRPWT" or "LLADTTHHRPWTGLGDTTHHRPWG" or "LLADTTHHRPWTGLGDTTHHRPWGLLADT" "THHRPWT" or "LLADTTHHRPWTGLGDTTHHRPWGLLADTTHHRPWTGLGDTTHHRP" "WGLLADTTHHRPWT" or "LLADTTHHRPWTGLGDTTHHRPWGLLADTTHHRPWTGLGD" <220> <221> MOD_RES <222> (13)..(132) <223> Continued from above: TTHHRPWGLLADTTHRPWTGLGDTTHRPWGLLADTTHRPWT" or "STADTSHHRPSTSGGESTHHRPSTSGGESSHHKPSTGSGDSSHHRPSGSSGESTHHKPST" or "VGADSTHHRPVTGAADTTHHRPVTAGADTTHHRPVTGGADTTHHRPATGGADTTHHRPGT" or "STADTSHHRPSLLADTTHHRPWTTSGGESTHHRPSVGADSTHHRPVTTSGGESSHHKPSG <220> <221> MOD_RES <222> (13)..(132) <223> Continued from the above: AADTTHHRPVTTGSGDSSHHRPSGSSGESTHHKPSTGGADTTHHR PAT" or "AAADTTHHRPWT" or "AAADTTHHRPWTAAADTTHHRPWTAAADTTHHRPWT AAADTTHHRPWTAAADTTHHRPWT" or "LLADAAHHRPWTLLADAAHHRPWTLLADAAHHR PWTLLADAAHHRPWTLLADAAHHRPWT" or "LLADTTAARPWTLLADTTAARPWTLLADTT <220> <221> MOD_RES <222> (13)..(132) <223> Continued from the above: AARPWTLLADTTAARPWTLLADTTAARPWT" or "LLADTTHHRPWTLLADTTHHRPWT" or "LLADTTHHRPWTLLADTTHHRPWTLLADTT HHRPWT" or "LLADTTHHRPWTLLADTTHHRPWTLLADTTHHRPWTLLADTTHHRPWT LLADTTHHRPWT" or "STSGSTVIGESTHHRPWSLIADSTHHSPWTILAESTHHKPWT <220> <221> MOD_RES <222> (13)..(132) <223> Continued from above: ILAETTHHRPWSIIGESSHHKPFTGLGDTTHHRPWGVLGDTT HHKPWTIVADSTHHRPWTGQVLPTTTPSSPSTTSGS" or "LLADTTHHRPWTVIGEST HHRPWSIIGESSHHKPFTGLGDTTHHRPWG" or "VIGESTHHRPWSIIGESSHHKPFT GLGDTTHHRPWGILAESTHHKPWT" <220> <223> For detailed descriptions of substitutions and preferred embodiments, reference is made to the submitted specification <400> 42 Leu Leu Ala Asp Thr Thr His His Arg Pro Trp Thr Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105 110 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 Xaa Xaa Xaa Xaa 130 <210> 43 <211> 132 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic polypeptide <220> <221> MOD_RES <222> (13)..(132) <223> This region may contain the following sequences: One: "LLADTTHHRPWT" or "VIGESTHHRPWS" or "LIADSTHHSPWT" Or "ILAESTHHKPWT" or "ILAETTHHRPWS" or "IIGESSHHKPFT" or "GLGDTTHHRPWG" or "VLGDTTHHKPWT" or "IVADSTHHRPWT" or <220> <221> MOD_RES <222> (13)..(132) <223> Continued from above: "STADTSHHRPS" or "TSGGESTHHRPS" or "TSGGESSHHKPS" or "TGSGDSSHHRPS" or "GSSGESTHHKPST" or "VGADSTHHRPVT" or "GAADTTHHRPVT" or "AGADTTHHRPVT" or "GGADTTHHRPAT" or "GGADTTHHRPGT" or <220> <221> MOD_RES <222> (13)..(132) <223> Continued from the above: "LLADTTHHRPWTVIGESTHHRPWS" or "LLADTTHHRPWTVIGESTHHRPWSIIGESSHHKPFT" or "LLADTTHHRPWTVIGESTHHRPWSIIGESSHHKPFTGLGDTTHHRPWGILAESTHHK PWT" or "LLADTTHHRPWTILAESTHHKPWT" or "LLADTTHHRPWTILAESTHHK <220> <221> MOD_RES <222> (13)..(132) <223> Continued from the above: PWTLLADTTHHRPWTILAESTHHKPWTLLADTTHHRPWT" or "LLADTTHHRPWTGLGDTTHHRPWG" or "LLADTTHHRPWTGLGDTTHHRPWGLLADT THHRPWT" or "LLADTTHHRPWTGLGDTTHHRPWGLLADTTHHRPWTGLGDTTHHRP WGLLADTTHHRPWT" or "LLADTTHHRPWTGLGDTTHHRPWGLLADTTHHRPWTGLGD <220> <221> MOD_RES <222> (13)..(132) <223> Continued from the above: TTHHRPWGLLADTTHHRPWTGLGDTTHHRPWGLLADTTHHRPWT" or "STADTSHHRPSTSGGESTHHRPSTSGGESSHHKPSTGSGDSSHHRPSGSSGESTHHKPST" or "VGADSTHHRPVTGAADTTHHRPVTAGADTTHHRPVTGGADTTHHRPATGGADTTHHRPGT" or "STADTSHHRPSLLADTTHHRPWTTSGGESTHHRPSVGADSTHHRPVTTSGGESSHHKPSG <220> <221> MOD_RES <222> (13)..(132) <223> Continued from above: AADTTHRPVTTGSGDSSHHRPSGSSGESTHHKPPSTGGADTTHHR PAT" or "AAADTTHHRPWT" or "AAADTTHHRPWTAAADTTHHRPWTAAADTTHHRPWT" AAADTTHHRPWTAAADTTHHRPWT" or "LLADAAHHRPWTLLADAAHHRPWTLLADAAHHR PWTLLADAAHHRPWTLLADAAHHRPWT" or "LLADTTAARPWTLLADTTAARPWTLLADTT <220> <221> MOD_RES <222> (13)..(132) <223> Continuing from above: AARPWTLLADTTAARPWTLLADTTAARPWT" or "LLADTTHHRPWTLLADTTHHRPWT" or "LLADTTHHRPWTLLADTTHHRPWTLLADTT HHRPWT" or "LLADTTHHRPWTLLADTTHHRPWTLLADTTHHRPWTLLADTTHHRPWT LLADTTHHRPWT" or "STSGSTVIGESTHHRPWSLIADSTHHSPWTILAESTHHKPWT" <220> <221> MOD_RES <222> (13)..(132) <223> Continued from above: ILAETTHHRPWSIIGESSHHKPFTGLGDTTHHRPWGVLGDTT HHKPWTIVADSTHHRPWTGQVLPTTTPSSPSTTSGS" or "LLADTTHHRPWTVIGEST HHRPWSIIGESSHHKPFTGLGDTTHHRPWG" or "VIGESTHHRPWSIIGESSHHKPFT GLGDTTHHRPWGILAESTHHKPWT" <220> <223> Detailed description of substitutions and preferred embodiments, See the submitted specification <400> 43 Val Ile Gly Glu Ser Thr His His Arg Pro Trp Ser Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105 110 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 Xaa Xaa Xaa Xaa 130 <210> 44 <211> 132 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic polypeptide <220> <221> MOD_RES <222> (13)..(132) <223> This region may contain the following sequences: One: "LLADTTHHRPWT" or "VIGESTHHRPWS" or "LIADSTHHSPWT" Or "ILAESTHHKPWT" or "ILAETTHHRPWS" or "IIGESSHHKPFT" or "GLGDTTHHRPWG" or "VLGDTTHHKPWT" or "IVADSTHHRPWT" or <220> <221> MOD_RES <222> (13)..(132) <223> Continued from above: "STADTSHHRPS" or "TSGGESTHHRPS" or "TSGGESSHHKPS" or "TGSGDSSHHRPS" or "GSSGESTHHKPST" or "VGADSTHHRPVT" or "GAADTTHHRPVT" or "AGADTTHHRPVT" or "GGADTTHHRPAT" or "GGADTTHHRPGT" or <220> <221> MOD_RES <222> (13)..(132) <223> Continuing from above: "LLADTTHHRPWTVIGESTHHRPWS" or "LLADTTHHRPWTVIGESTHHRPWSIIGESSHHKPFT" or "LLADTTHHRPWTVIGESTHHRPWSIIGESSHHKPFTGLGDTTHRPWGILAESTHHK PWT or "LLADTTHHRPWTILAESTHHKPWT" or "LLADTTHHRPWTILAESTHHK" <220> <221> MOD_RES <222> (13)..(132) <223> Continued from above: PWTLLADTTHHRPWTILAESTHHKPWTLLADTTHHRPWT" or "LLADTTHHRPWTGLGDTTHHRPWG" or "LLADTTHHRPWTGLGDTTHHRPWGLLADT" "THHRPWT" or "LLADTTHHRPWTGLGDTTHHRPWGLLADTTHHRPWTGLGDTTHHRP" "WGLLADTTHHRPWT" or "LLADTTHHRPWTGLGDTTHHRPWGLLADTTHHRPWTGLGD" <220> <221> MOD_RES <222> (13)..(132) <223> Continued from above: TTHHRPWGLLADTTHRPWTGLGDTTHRPWGLLADTTHRPWT" or "STADTSHHRPSTSGGESTHHRPSTSGGESSHHKPSTGSGDSSHHRPSGSSGESTHHKPST" or "VGADSTHHRPVTGAADTTHHRPVTAGADTTHHRPVTGGADTTHHRPATGGADTTHHRPGT" or "STADTSHHRPSLLADTTHHRPWTTSGGESTHHRPSVGADSTHHRPVTTSGGESSHHKPSG <220> <221> MOD_RES <222> (13)..(132) <223> Continued from the preceding: AADTTHHRPVTTGSGDSSHHRPSGSSGESTHHKPSTGGADTTHHR PAT" or "AAADTTHHRPWT" or "AAADTTHHRPWTAAADTTHHRPWTAAADTTHHRPWT AAADTTHHRPWTAAADTTHHRPWT" or "LLADAAHHRPWTLLADAAHHRPWTLLADAAHHR PWTLLADAAHHRPWTLLADAAHHRPWT" or "LLADTTAARPWTLLADTTAARPWTLLADTT <220> <221> MOD_RES <222> (13)..(132) <223> Continued from the preceding: AARPWTLLADTTAARPWTLLADTTAARPWT" or "LLADTTHHRPWTLLADTTHHRPWT" or "LLADTTHHRPWTLLADTTHHRPWTLLADTT HHRPWT" or "LLADTTHHRPWTLLADTTHHRPWTLLADTTHHRPWTLLADTTHHRPWT LLADTTHHRPWT" or "STSGSTVIGESTHHRPWSLIADSTHHSPWTILAESTHHKPWT <220> <221> MOD_RES <222> (13)..(132) <223> Continued from the preceding: ILAETTHHRPWSIIGESSHHKPFTGLGDTTHHRPWGVLGDTT HHKPWTIVADSTHHRPWTGQVLPTTTPSSPSTTSGS" or "LLADTTHHRPWTVIGEST HHRPWSIIGESSHHKPFTGLGDTTHHRPWG" or "VIGESTHHRPWSIIGESSHHKPFT GLGDTTHHRPWGLAESTHKPWT" <220> <223> See more of the news updates, see Remove the mouse. <400> 44 I'm Only Glue To Be Thr His His Lys Pro Trp Thr Nothing Nothing Nothing 1 5 10 15 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes 20 25 30 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes 35 40 45 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes 50 55 60 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes 65 70 75 80 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes 85 90 95 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes 100 105 110 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes 115 120 125 Blood Blood Blood Blood! 130 <210> 45 <211> 132 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic polypeptide <220> <221> MOD_RES <222> (13)..(132) <223> This region may contain the following sequences: One: "LLADTTHHRPWT" or "VIGESTHHRPWS" or "LIADSTHHSPWT" Or "ILAESTHHKPWT" or "ILAETTHHRPWS" or "IIGESSHHKPFT" or "GLGDTTHHRPWG" or "VLGDTTHHKPWT" or "IVADSTHHRPWT" or <220> <221> MOD_RES <222> (13)..(132) <223> Continued from above: "STADTSHHRPS" or "TSGGESTHHRPS" or "TSGGESSHHKPS" or "TGSGDSSHHRPS" or "GSSGESTHHKPST" or "VGADSTHHRPVT" or "GAADTTHHRPVT" or "AGADTTHHRPVT" or "GGADTTHHRPAT" or "GGADTTHHRPGT" or <220> <221> MOD_RES <222> (13)..(132) <223> Continuing from above: "LLADTTHHRPWTVIGESTHHRPWS" or "LLADTTHHRPWTVIGESTHHRPWSIIGESSHHKPFT" or "LLADTTHHRPWTVIGESTHHRPWSIIGESSHHKPFTGLGDTTHRPWGILAESTHHK PWT" or "LLADTTHHRPWTILAESTHHKPWT" or "LLADTTHHRPWTILAESTHHK <220> <221> MOD_RES <222> (13)..(132) <223> Continued from the above: PWTLLADTTHHRPWTILAESTHHKPWTLLADTTHHRPWT" or "LLADTTHHRPWTGLGDTTHHRPWG" or "LLADTTHHRPWTGLGDTTHHRPWGLLADT THHRPWT" or "LLADTTHHRPWTGLGDTTHHRPWGLLADTTHHRPWTGLGDTTHHRP WGLLADTTHHRPWT" or "LLADTTHHRPWTGLGDTTHHRPWGLLADTTHHRPWTGLGD <220> <221> MOD_RES <222> (13)..(132) <223> Continued from the above: TTHHRPWGLLADTTHHRPWTGLGDTTHHRPWGLLADTTHHRPWT" or "STADTSHHRPSTSGGESTHHRPSTSGGESSHHKPSTGSGDSSHHRPSGSSGESTHHKPST" or "VGADSTHHRPVTGAADTTHHRPVTAGADTTHHRPVTGGADTTHHRPATGGADTTHHRPGT" or "STADTSHHRPSLLADTTHHRPWTTSGGESTHHRPSVGADSTHHRPVTTSGGESSHHKPSG <220> <221> MOD_RES <222> (13)..(132) <223> Continued from the above: AADTTHHRPVTTGSGDSSHHRPSGSSGESTHHKPSTGGADTTHHR PAT" or "AAADTTHHRPWT" or "AAADTTHHRPWTAAADTTHHRPWTAAADTTHHRPWT" AAADTTHHRPWTAAADTTHHRPWT" or "LLADAAHHRPWTLLADAAHHRPWTLLADAAHHR PWTLLADAAHHRPWTLLADAAHHRPWT" or "LLADTTAARPWTLLADTTAARPWTLLADTT <220> <221> MOD_RES <222> (13)..(132) <223> Continuing from above: AARPWTLLADTTAARPWTLLADTTAARPWT" or "LLADTTHHRPWTLLADTTHHRPWT" or "LLADTTHHRPWTLLADTTHHRPWTLLADTT HHRPWT" or "LLADTTHHRPWTLLADTTHHRPWTLLADTTHHRPWTLLADTTHHRPWT LLADTTHHRPWT" or "STSGSTVIGESTHHRPWSLIADSTHHSPWTILAESTHHKPWT" <220> <221> MOD_RES <222> (13)..(132) <223> Continued from above: ILAETTHHRPWSIIGESSHHKPFTGLGDTTHHRPWGVLGDTT HHKPWTIVADSTHHRPWTGQVLPTTTPSSPSTTSGS" or "LLADTTHHRPWTVIGEST" HHRPWSIIGESSHHKPFTGLGDTTHHRPWG" or "VIGESTHHRPWSIIGESSHHKPFT" GLGDTTHHRPWGILAESTHHKPWT" <220> <223> Detailed description of alternative and preferred embodiments, See the submitted instruction manual. <400> 45 Ile Ile Gly Glu Ser Ser His His Lys Pro Phe Thr Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105 110 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 Xaa Xaa Xaa Xaa 130 <210> 46 <211> 132 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: Synthetic Polypeptide <220> <221> MOD_RES <222> (13)..(132) <223> This region may contain the following sequences: One: "LLADTTHHRPWT" or "VIGESTHHRPWS" or "LIADSTHHSPWT" Or "ILAESTHHKPWT" or "ILAETTHHRPWS" or "IIGESSHHKPFT" or "GLGDTTHHRPWG" or "VLGDTTHHKPWT" or "IVADSTHHRPWT" or <220> <221> MOD_RES <222> (13)..(132) <223> Continued from above: "STADTSHHRPS" or "TSGGESTHHRPS" or "TSGGESSHHKPS" or "TGSGDSSHHRPS" or "GSSGESTHHKPST" or "VGADSTHHRPVT" or "GAADTTHHRPVT" or "AGADTTHHRPVT" or "GGADTTHHRPAT" or "GGADTTHHRPGT" or <220> <221> MOD_RES <222> (13)..(132) <223> Continuing from above: "LLADTTHHRPWTVIGESTHHRPWS" or "LLADTTHHRPWTVIGESTHHRPWSIIGESSHHKPFT" or "LLADTTHHRPWTVIGESTHHRPWSIIGESSHHKPFTGLGDTTHRPWGILAESTHHK PWT or "LLADTTHHRPWTILAESTHHKPWT" or "LLADTTHHRPWTILAESTHHK" <220> <221> MOD_RES <222> (13)..(132) <223> Continued from above: PWTLLADTTHHRPWTILAESTHHKPWTLLADTTHHRPWT" or "LLADTTHHRPWTGLGDTTHHRPWG" or "LLADTTHHRPWTGLGDTTHHRPWGLLADT" "THHRPWT" or "LLADTTHHRPWTGLGDTTHHRPWGLLADTTHHRPWTGLGDTTHHRP" "WGLLADTTHHRPWT" or "LLADTTHHRPWTGLGDTTHHRPWGLLADTTHHRPWTGLGD" <220> <221> MOD_RES <222> (13)..(132) <223> Continued from above: TTHHRPWGLLADTTHRPWTGLGDTTHRPWGLLADTTHRPWT" or "STADTSHHRPSTSGGESTHHRPSTSGGESSHHKPSTGSGDSSHHRPSGSSGESTHHKPST" or "VGADSTHHRPVTGAADTTHHRPVTAGADTTHHRPVTGGADTTHHRPATGGADTTHHRPGT" or "STADTSHHRPSLLADTTHHRPWTTSGGESTHHRPSVGADSTHHRPVTTSGGESSHHKPSG <220> <221> MOD_RES <222> (13)..(132) <223> Continued from above: AADTTHRPVTTGSGDSSHHRPSGSSGESTHHKPPSTGGADTTHHR PAT" or "AAADTTHHRPWT" or "AAADTTHHRPWTAAADTTHHRPWTAAADTTHHRPWT" AAADTTHHRPWTAAADTTHHRPWT" or "LLADAAHHRPWTLLADAAHHRPWTLLADAAHHR PWTLLADAAHHRPWTLLADAAHHRPWT" or "LLADTTAARPWTLLADTTAARPWTLLADTT <220> <221> MOD_RES <222> (13)..(132) <223> Continuing from above: AARPWTLLADTTAARPWTLLADTTAARPWT" or "LLADTTHHRPWTLLADTTHHRPWT" or "LLADTTHHRPWTLLADTTHHRPWTLLADTT HHRPWT" or "LLADTTHHRPWTLLADTTHHRPWTLLADTTHHRPWTLLADTTHHRPWT LLADTTHHRPWT" or "STSGSTVIGESTHHRPWSLIADSTHHSPWTILAESTHHKPWT" <220> <221> MOD_RES <222> (13)..(132) <223> Continued from above: ILAETTHHRPWSIIGESSHHKPFTGLGDTTHHRPWGVLGDTT HHKPWTIVADSTHHRPWTGQVLPTTTPSSPSTTSGS" or "LLADTTHHRPWTVIGEST" HHRPWSIIGESSHHKPFTGLGDTTHHRPWG" or "VIGESTHHRPWSIIGESSHHKPFT" GLGDTTHHRPWGILAESTHHKPWT" <220> <223> Detailed description of alternative and preferred embodiments, See the submitted instruction manual. <400> 46 Gly Leu Gly Asp Thr Thr His His Arg Pro Trp Gly Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105 110 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 Xaa Xaa Xaa Xaa 130 <210> 47 <211> 24 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: synthetic peptide <220> <221> MOD_RES <222> (13)..(24) <223> This region may comprise the following sequences One: "VIGESTHHRPWS" or "ILAESTHHKPWT" or "IIGESSHHKPFT" Or "GLGDTTHHRPWG" <220> <223> Detailed description of alternative and preferred embodiments, See the submitted instruction manual. <400> 47 Leu Leu Ala Asp Thr Thr His His Arg Pro Trp Thr Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 <210> 48 <211> twenty four <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <220> <221> MOD_RES <222> (13) (24) <223> This region may contain the following sequences: One: "LLADTTHHRPWT" or "ILAESTHHKPWT" or "IIGESSHHKPFT" Or "GLGDTTHHRPWG" <220> <223> Detailed description of alternative and preferred embodiments, See the submitted instruction manual. <400> 48 Val Ile Gly Glu Ser Thr His His Arg Pro Trp Ser Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 <210> 49 <211> twenty four <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <220> <221> MOD_RES <222> (13) (24) <223> This region may contain the following sequences: One: "LLADTTHHRPWT" or "VIGESTHHRPWS" or "IIGESSHHKPFT" Or "GLGDTTHHRPWG" <220> <223> Detailed description of alternative and preferred embodiments, See the submitted instruction manual. <400> 49 Ile Leu Ala Glu Ser Thr His His Lys Pro Trp Thr Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 <210> 50 <211> twenty four <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <220> <221> MOD_RES <222> (13) (24) <223> This region may contain the following sequences: One: "LLADTTHHRPWT" or "ILAESTHHKPWT" or "VIGESTHHRPWS" Or "GLGDTTHHRPWG" <220> <223> Detailed description of alternative and preferred embodiments, See the submitted instruction manual. <400> 50 Ile Ile Gly Glu Ser Ser His His Lys Pro Phe Thr Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 <210> 51 <211> twenty four <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <220> <221> MOD_RES <222> (13) (24) <223> This region may contain the following sequences: One: "LLADTTHHRPWT" or "ILAESTHHKPWT" or "IIGESSHHKPFT" Or "VIGESTHHRPWS" <220> <223> Detailed description of alternative and preferred embodiments, See the submitted instruction manual. <400> 51 Gly Leu Gly Asp Thr Thr His His Arg Pro Trp Gly Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 <210> 52 <211> 36 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic polypeptide <220> <221> MOD_RES <222> (13) (36) <223> This region may contain the following sequences: Two options: "VIGESTHHRPWS" or "ILAESTHHKPWT" or "IIGESSHHKPFT" Or "GLGDTTHHRPWG" <220> <223> Detailed description of alternative and preferred embodiments, See the submitted instruction manual. <400> 52 Leu Leu Ala Asp Thr Thr His His Arg Pro Trp Thr Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa 35 <210> 53 <211> 36 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic polypeptide <220> <221> MOD_RES <222> (13) (36) <223> This region may contain the following sequences: Two options: "LLADTTHHRPWT" or "ILAESTHHKPWT" or "IIGESSHHKPFT" Or "GLGDTTHHRPWG" <220> <223> Detailed description of alternative and preferred embodiments, See the submitted instruction manual. <400> 53 Val Ile Gly Glu Ser Thr His His Arg Pro Trp Ser Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa 35 <210> 54 <211> 36 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic polypeptide <220> <221> MOD_RES <222> (13) (36) <223> This region may contain the following sequences: Two options: "LLADTTHHRPWT" or "VIGESTHHRPWS" or "IIGESSHHKPFT" Or "GLGDTTHHRPWG" <220> <223> Detailed description of alternative and preferred embodiments, See the submitted instruction manual. <400> 54 Ile Leu Ala Glu Ser Thr His His Lys Pro Trp Thr Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa 35 <210> 55 <211> 36 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic polypeptide <220> <221> MOD_RES <222> (13) (36) <223> This region may contain the following sequences: Two options: "LLADTTHHRPWT" or "ILAESTHHKPWT" or "VIGESTHHRPWS" Or "GLGDTTHHRPWG" <220> <223> Detailed description of alternative and preferred embodiments, See the submitted instruction manual. <400> 55 Ile Ile Gly Glu Ser Ser His His Lys Pro Phe Thr Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa 35 <210> 56 <211> 36 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic polypeptide <220> <221> MOD_RES <222> (13) (36) <223> This region may contain the following sequences: Two options: "LLADTTHHRPWT" or "ILAESTHHKPWT" or "IIGESSHHKPFT" Or "VIGESTHHRPWS" <220> <223> Detailed description of alternative and preferred embodiments, See the submitted instruction manual. <400> 56 Gly Leu Gly Asp Thr Thr His His Arg Pro Trp Gly Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa 35 <210> 57 <211> 48 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: synthetic polypeptide <220> <221> MOD_RES <222> (13)..(48) <223> This region may comprise three of the following sequences : "VIGESTHHRPWS" or "ILAESTHHKPWT" or "IIGESSHHKPFT" or "GLGDTTHHRPWG" <220> <223> For a detailed description of substitutions and preferred embodiments, reference is made to the submitted specification <400> 57 Leu Leu Ala Asp Thr Thr His His Arg Pro Trp Thr Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 <210> 58 <211> 48 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic polypeptide <220> <221> MOD_RES <222> (13) (48) <223> This region may contain the following sequences: Three options: "LLADTTHHRPWT", "ILAESTHHKPWT", or "IIGESSHHKPFT". Or "GLGDTTHHRPWG" <220> <223> Detailed description of alternative and preferred embodiments, See the submitted instruction manual. <400> 58 Val Ile Gly Glu Ser Thr His His Arg Pro Trp Ser Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 <210> 59 <211> 48 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic polypeptide <220> <221> MOD_RES <222> (13) (48) <223> This region may contain the following sequences: Three: "LLADTTHHRPWT" or "VIGESTHHRPWS" or "IIGESSHHKPFT" Or "GLGDTTHHRPWG" <220> <223> Detailed description of alternative and preferred embodiments, See the submitted instruction manual. <400> 59 Ile Leu Ala Glu Ser Thr His His Lys Pro Trp Thr Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 <210> 60 <211> 48 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic polypeptide <220> <221> MOD_RES <222> (13) (48) <223> This region may contain the following sequences: Three "LLADTTHHRPWT" or "ILAESTHHKPWT" or "VIGESTHHRPWS" Or "GLGDTTHHRPWG" <220> <223> Detailed description of alternative and preferred embodiments, See the submitted instruction manual. <400> 60 Ile Ile Gly Glu Ser Ser His His Lys Pro Phe Thr Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 <210> 61 <211> 48 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: synthetic Polypeptide <220> <221> MOD_RES <222> (13)..(48) <223> This region may comprise three of the following sequences : "LLADTTHHRPWT" or "ILAESTHHKPWT" or "IIGESSHHKPFT" or "VIGESTHHRPWS" <220> <223> For a detailed description of substitutions and preferred embodiments , refer to the submitted specification <400> 61 Gly Leu Gly Asp Thr Thr His His Arg Pro Trp Gly Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 <210> 62 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 62 Ala Cys Ala Pro Leu Met Phe Ser Gln Cys 1 5 10 <210> 63 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 63 Ala Cys His Ala Ser Leu Lys His Arg Cys 1 5 10 <210> 64 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 64 Ala Cys Leu Ser Thr Lys Thr Asn Ile Cys 1 5 10 <210> 65 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 65 Ala Cys Thr Thr Pro Ser Lys His Gln Cys 1 5 10 <210> 66 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 66 Ala His Phe Ser Pro Asn Leu Leu Leu Gly Gly 1 5 10 <210> 67 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 67 Ala His Ser Leu Lys Ser Ile Thr Asn His Gly Leu 1 5 10 <210> 68 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 68 Ala Lys Gln Thr Val Pro Val 1 5 <210> 69 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 69 Ala Lys Thr Leu Met Pro Ser Pro Phe Pro Arg Thr 1 5 10 <210> 70 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 70 Ala Met Ser Gln Thr Met Thr Ala Ala Ile Glu Lys 1 5 10 <210> 71 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 71 Ala Asn Pro Pro Leu Ser Leu 1 5 <210> 72 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 72 Ala Asn Pro Tyr His Arg His 1 5 <210> 73 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 73 Ala Pro Leu Ser Leu Ser Leu 1 5 <210> 74 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 74 Ala Pro Tyr His Pro Thr Ile Pro Ala Ser Val His Gly Gly Gly Lys 1 5 10 15 <210> 75 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <220> <221> MOD_RES <222> (12)..(12) <223> Any amino acid <400> 75 Ala Ser Ala Val Gly Ser Leu Ser Ile Arg Trp Xaa 1 5 10 <210> 76 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 76 Ala Ser Gly Pro Thr Asn Val 1 5 <210> 77 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 77 Ala Ser His Asn Pro Lys Leu 1 5 <210> 78 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 78 Ala Ser Trp Val Asp Ser Arg Gln Pro Ser Ala Ala 1 5 10 <210> 79 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 79 Ala Thr Phe Ser Pro Pro Leu 1 5 <210> 80 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 80 Ala Thr Trp Ser His His Leu Ser Ser Ala Gly Leu 1 5 10 <210> 81 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 81 Ala Thr Trp Ser His His Leu Ser Ser Ala Gly Leu Gly Gly Gly Ser 1 5 10 15 <210> 82 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 82 Cys Ala His Leu Ser Pro His Lys Cys 1 5 <210> 83 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 83 Cys Asp Ile Pro Trp Arg Asn Glu Cys 1 5 <210> 84 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 84 Cys Asp Pro Leu Arg Gln His Ser Cys 1 5 <210> 85 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 85 Cys Asp Ser Leu Gly His Trp Leu Cys 1 5 <210> 86 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 86 Cys Asp Tyr Thr Thr Arg His Ser Cys 1 5 <210> 87 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 87 Cys His Gly Thr Leu Asn Pro Glu Cys 1 5 <210> 88 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 88 Cys His His Asn Leu Ser Trp Glu Cys 1 5 <210> 89 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 89 Cys His Ile Trp Thr Leu Ala Ser Cys 1 5 <210> 90 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 90 Cys His Asn Thr Phe Ser Pro Arg Cys 1 5 <210> 91 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 91 Cys Ile Pro Leu His Ala Ser Leu Cys 1 5 <210> 92 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 92 Cys Ile Thr Thr Thr Ser Leu Ser Cys 1 5 <210> 93 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 93 Cys Lys Leu Thr Thr Cys Lys Asp Cys 1 5 <210> 94 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 94 Cys Lys Asn His Thr Thr Phe Trp Cys 1 5 <210> 95 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 95 Cys Leu Lys Leu Leu Ser Arg Ser Cys 1 5 <210> 96 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 96 Cys Leu Leu Lys Ala His Pro Ser Cys 1 5 <210> 97 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 97 Cys Leu Asn Gln Leu Lys Gln Ala Cys 1 5 <210> 98 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 98 Cys Leu Ser Thr Lys Thr Asn Ile Cys 1 5 <210> 99 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 99 Cys Met Asn Phe Pro Ser Pro His Cys 1 5 <210> 100 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 100 Cys Asn Tyr Pro Thr Leu Lys Ser Cys 1 5 <210> 101 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 101 Cys Pro Gln Leu Thr Val Gly Gln His Arg Thr 1 5 10 <210> 102 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 102 Cys Pro Gln Ser Pro Thr Tyr Thr Cys 1 5 <210> 103 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 103 Cys Pro Ser Ser Ala Ile His Thr Cys 1 5 <210> 104 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 104 Cys Pro Thr Ser Thr Ala Arg Ile Cys 1 5 <210> 105 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 105 Cys Gln Ala Ser Ser Phe Pro Ser Cys 1 5 <210> 106 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 106 Cys Gln Pro Tyr Phe Trp Tyr Arg Cys 1 5 <210> 107 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 107 Cys Gln Thr Leu Thr Pro Ser Ile Cys 1 5 <210> 108 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 108 Cys Ser Lys Leu Gly His Leu Trp Cys 1 5 <210> 109 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <220> <221> MOD_RES <222> (9)..(9) <223> Any amino acid <400> 109 Cys Ser Lys Thr Pro Glu Arg Ile Xaa 1 5 <210> 110 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 110 Cys Ser Asn Asn Asn Arg Met Thr Cys 1 5 <210> 111 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 111 Cys Ser Pro Ile Leu Ser Leu Ser Cys 1 5 <210> 112 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 112 Cys Ser Pro Thr Asn Phe Thr Arg Cys 1 5 <210> 113 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 113 Cys Ser Arg Pro Ala Met Asn Val Cys 1 5 <210> 114 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 114 Cys Ser Thr Lys Ala Tyr Pro Asn Cys 1 5 <210> 115 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 115 Cys Ser Thr Ser Ser Cys Gly Ser Cys 1 5 <210> 116 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 116 Cys Ser Tyr Trp Gly His Arg Asp Cys 1 5 <210> 117 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 117 Cys Thr Ala His Asp Ala Asn Ala Cys 1 5 <210> 118 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 118 Cys Thr Ala Asn Ser Glu Lys Thr Cys 1 5 <210> 119 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 119 Cys Thr His Pro Lys Ala Ser Met Cys 1 5 <210> 120 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 120 Cys Thr Lys Thr Ile Asn Gly Lys Cys 1 5 <210> 121 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 121 Cys Thr Asn Met Gln Ser Pro Leu Cys 1 5 <210> 122 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 122 Cys Thr Pro Phe Thr Lys Leu Pro Cys 1 5 <210> 123 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 123 Cys Thr Pro Thr Thr Asp Ser Ile Cys 1 5 <210> 124 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 124 Cys Thr Gln Gln Asn Gly His Pro Cys 1 5 <210> 125 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 125 Cys Thr Thr Pro Ser Lys His Gln Cys 1 5 <210> 126 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 126 Cys Thr Tyr Asn Val Ala Lys Pro Cys 1 5 <210> 127 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 127 Asp Lys Leu His Arg Leu Ala 1 5 <210> 128 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 128 Asp Leu Asn Tyr Phe Thr Leu Ser Ser Lys Arg Glu 1 5 10 <210> 129 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 129 Asp Leu Pro Pro Thr Leu His Thr Thr Gly Ser Pro 1 5 10 <210> 130 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 130 Asp Met Arg Gln Gln Arg Ser 1 5 <210> 131 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 131 Asp Gln Tyr Trp Gly Leu Arg 1 5 <210> 132 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 132 Asp Ser Ser Asn Pro Ile Phe Trp Arg Pro Ser Ser 1 5 10 <210> 133 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 133 Asp Ser Ser Asn Pro Ile Phe Trp Arg Pro Ser Ser Gly Gly Gly Ser 1 5 10 15 <210> 134 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 134 Glu Phe Leu Gly Val Pro Ala Ser Leu Val Asn Pro 1 5 10 <210> 135 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 135 Glu Pro Asn His Thr Arg Phe 1 5 <210> 136 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 136 Glu Pro Arg Arg Ala Val Ala Ala Leu 1 5 <210> 137 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 137 Glu Pro Arg Arg Ala Val Ala Glu Leu 1 5 <210> 138 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 138 Glu Pro Arg Arg Glu Val Ala Glu Leu 1 5 <210> 139 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 139 Glu Pro Arg Arg Glu Val Cys Glu Leu 1 5 <210> 140 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 140 Glu Ser Asp Leu Thr His Ala Leu His Trp Leu Gly 1 5 10 <210> 141 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 141 Glu Ser Leu Lys Ser Ile Ser 1 5 <210> 142 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 142 Glu Thr Arg Thr Gln Leu Leu 1 5 <210> 143 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 143 Glu Thr Val Cys Ala Ser Ser 1 5 <210> 144 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 144 Glu Thr Tyr Ala Arg Pro Leu 1 5 <210> 145 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 145 Glu Thr Tyr Gln Gln Pro Leu 1 5 <210> 146 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 146 Glu Val His Ser Thr Asp Arg Tyr Arg Ser Ile Pro 1 5 10 <210> 147 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 147 Phe Gly Leu Gln Pro Thr Gly Asp Ile Ala Arg Arg 1 5 10 <210> 148 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 148 Phe Ser Met Asp Asp Pro Glu Arg Val Arg Ser Pro 1 5 10 <210> 149 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 149 Phe Ser Pro Leu His Thr Ser Thr Tyr Arg Pro Ser 1 5 10 <210> 150 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 150 Phe Thr Leu Pro Thr Ile Arg 1 5 <210> 151 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 151 Phe Val Asn Leu Leu Gly Gln 1 5 <210> 152 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 152 Gly Asp Phe Asn Ser Gly His His Thr Thr Thr Arg 1 5 10 <210> 153 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 153 Gly Gly Gly Ala Ala Ala Ala 1 5 <210> 154 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 154 Gly Ile His Val Pro Trp Met Pro Pro Val Ala Phe 1 5 10 <210> 155 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 155 Gly Ile His Val Pro Trp Met Pro Pro Val Ala Phe Gly Gly Gly Ser 1 5 10 15 <210> 156 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 156 Gly Pro Ser Asn Asn ...

Claims

1. An apparatus comprising a three-dimensional structure and a therapeutic agent, wherein the three-dimensional structure comprises 50% to 90% by weight calcium phosphate and 10% to 50% by weight a polymer, wherein the three-dimensional structure has a plurality of micropores, and wherein the average pore size of the micropores is 50 micrometers to 250 micrometers, and wherein the therapeutic agent comprises a bone morphogenetic protein and a targeting portion, wherein the targeting portion is non-covalently bound to the calcium phosphate, and wherein the targeting portion comprises SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 7, or a combination thereof.

2. The device of claim 1, wherein the micropores provide additional surface area for the three-dimensional structure to contact the therapeutic agent.

3. The apparatus of claim 1, wherein the three-dimensional structure is generated by three-dimensional printing of a formulation comprising the calcium phosphate, the polymer, and polyethylene glycol, wherein the micropores are formed after the polyethylene glycol is removed from the three-dimensional structure.

4. The apparatus of claim 1, wherein the micropores are formed by releasing carbon dioxide from a foaming agent present during the fabrication of the three-dimensional structure.

5. The apparatus of claim 4, wherein the foaming agent comprises sodium bicarbonate.

6. The apparatus according to claim 1, wherein the open porosity of the three-dimensional structure is 15% to 50%, or the density of the three-dimensional structure is 1 g / cm³. 3 Up to 2 g / cm 3 Alternatively, the diameter of the support pillars of the three-dimensional structure may be between 300 and 600 micrometers.

7. The apparatus of claim 1, wherein the polymer comprises polycaprolactone, caprolactone / glycolic acid copolymer, poly(D,L-lactide-co-glycolic acid) copolymer, or p-dioxanone / L-lactide copolymer, or a combination thereof.

8. The apparatus of claim 1, wherein the three-dimensional structure is formed from filament using an extrusion-based three-dimensional printing method.

Images