Bone composite material and composition for preparing bone composite material

Abstract

Disclosed herein are multi-component or multi-part compositions for preparing bone constructs for, for example, trauma or cancer patients. The multi-part compositions are innovative combinations based on fibrinogen, thrombin, hydrogels, and calcium / phosphorus salts. Advantageously, the multi-part compositions can be printed to produce bone constructs using a 3D printing process to produce accurate and precise bone constructs with desired geometric shapes.

Description

Field of the Invention

[0001] The present invention relates to tissue manufacturing. In particular, compositions for 3D printing of bone composites are disclosed herein. These compositions allow bone composites to be printed from a design with accuracy and fidelity. The present invention also relates to methods for 3D printing bone composites, and bone composites obtainable from the compositions disclosed herein.

[0002] background

[0003] Bone tissue grafts are widely used in plastic surgery, neurosurgery, maxillofacial surgery, and dental surgery. Although effective, the use of autologous bone grafts and allografts has many limitations.

[0004] Autologous bone grafts are harvested from the patient, and therefore they require additional surgery and have increased risks associated with their harvesting, such as risk of infection, blood loss, and compromised structural integrity at the donor site.

[0005] Alternatively, load-bearing allograft bone can be used as a substitute for autologous bone. Primarily, they are extracted from cadavers and avoid the complexity and patient exposure associated with obtaining autologous bone from living donors. However, issues surrounding sterility, suitability, and long-term supply remain.

[0006] Prior to use, both allograft bone tissue and autograft bone tissue need to be shaped for a specific application specified by a surgeon. Naturally, considerations regarding the sterility of the shaped bone graft are paramount. In some cases, implantation of feasible bone graft substitutes is limited by the available shapes and sizes extracted from living or deceased donors.

[0007] Synthetic bone substitute materials and bone chips provide a more flexible raw material and can be easily reshaped and reshaped, however, they do not immediately provide mechanical support to the patient. Such materials are often used to fill oddly shaped bone defects, however, they are not well suited for wrapping or resurfacing bone.

[0008] The use of human allograft bone tissue is very common in orthopedic surgery. Invariably, the allograft tissue serves as a scaffold for a composition comprising a material having osteoconductive, osteoinductive and / or osteogenic properties. Suitable materials include proteins and / or stem cells.

[0009] International Patent Publication No. WO2012118843 addresses the above mentioned drawbacks of allogeneic and autologous bone grafts by providing a biocompatible modular scaffold optionally coated with, inter alia, bone morphogenetic protein.

[0010] In the prior art, 3D printing technology and bio-inks have also been proposed as an alternative to improve the defects of traditional allogeneic bone grafts and autologous bone grafts in bone replacement therapy. For example, International Patent Publication No. WO2018078130 discloses a printable bio-ink comprising cellulose nanofibrils, calcium-containing particles and living cells such as mesenchymal stem cells, osteoblasts or induced pluripotent stem cells. Cellulose nanofibrils are crucial for the structural integrity of bone constructs.

[0011] Despite the state of the art, there is still a need for additional alternatives to the traditional allogeneic bone grafts and autologous bone grafts used in bone replacement therapy. Such alternatives should be able to be manufactured under controlled, sterile manufacturing conditions. Crucial to this is the ability of the material to be shaped in a precise and accurate manner according to the specifications of the surgeon or other health professional. Naturally, all such alternatives should exhibit mechanical integrity and be able to provide structural support and load-bearing support after transplantation. Ideally, the material should be biocompatible and non-toxic to bioactive materials having osteoconductive properties, osteoinductive properties and / or osteogenic properties. SUMMARY OF THE INVENTION

[0013] When used herein with reference to the present invention, the words "comprises / comprising" and the words "having / including" are used to specify the presence of stated features, integers, steps or components, but do not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

[0014] It should be understood by those skilled in the art that the specific embodiments disclosed herein should not be read in isolation, and that the embodiments disclosed in this specification are intended to be read in combination with each other, rather than individually. Therefore, each embodiment can be used as a basis for modifying or limiting other embodiments disclosed herein.

[0015] Concentration, amount and other numerical data can be described or presented in range format in this article.It should be understood that such range format is used only for convenience and simplicity, and therefore should be flexibly interpreted as not only including the numerical value clearly described as the limit of range, but also including all independent numerical values ​​or sub-ranges covered in the range, as each numerical value and sub-range are clearly described.As an illustration, the numerical range of "10 to 100" should be interpreted as not only including the value clearly described from 10 to 100, but also including independent values ​​and sub-ranges in the indicated range.Therefore, included in the numerical range are independent values ​​such as 10,11,12,13, ..., 97,98,99,100 and such as from 10 to 40, from 25 to 40 and 50 to 60 sub-ranges, etc. This same principle is applicable to the scope of only narrating a numerical value, such as "at least 10".In addition, no matter how the breadth of the scope or the characteristic of the description, such an explanation should be applied.

[0016] Compositions of the present invention

[0017] In a first aspect, the present invention provides a multi-part composition for three-dimensional printing of a bone composite material, the multi-part composition comprising:

[0018] i) a first part comprising fibrinogen in a pharmaceutically acceptable carrier;

[0019] ii) a second part comprising thrombin in a pharmaceutically acceptable carrier; and

[0020] iii) a third part comprising a pharmaceutically acceptable hydrogel mixed with at least one biocompatible inorganic material, the at least one inorganic material providing a source of at least one of calcium atoms and phosphorus atoms,

[0021] wherein the third part has an apparent viscosity selected from the group consisting of:

[0022] The viscosity of the sample was measured by a rotational viscometer at 25°C and a pressure of 1 atm in 0.1 s. -1 A shear rate of from about 1 Pa·s to about 300 Pa·s, and

[0023] The viscosity of the sample was measured by a rotational viscometer at 25 °C and a pressure of 1 atm in 1 s. -1 The shear rate is from about 1 Pa·s to about 100 Pa·s,

[0024] And further wherein the third part is a separate composition, or the third part is mixed with the first part or the second part of the multi-part composition, such that at least the first part and the second part of the multi-part composition are not mixed prior to printing by the three-dimensional printing device.

[0025] As used herein, the term rotational viscometer refers to an apparatus that works on the principle of measuring the force (torque) acting on a rotor when it rotates at a constant angular velocity (rotational speed) in a liquid. A rotational viscometer is used to measure the viscosity of a Newtonian liquid (shear-independent viscosity) or a non-Newtonian liquid (shear-dependent viscosity or apparent viscosity). The third part of the multi-part composition of the present invention is shear-thinning, i.e., they have a shear-dependent viscosity; therefore, it is necessary to specify the shear rate when measuring the viscosity. For additional information, the technician refers to ASTM D2196-18e1: Standard Test Methods for Rheological Properties of Non-Newtonian Materials by Rotational Viscometer, the contents of which are incorporated herein by reference.

[0026] In one embodiment, the present invention provides a two-part composition wherein:

[0027] The first part (fibrinogen) is mixed with the third part, or

[0028] The second part (thrombin) is mixed with the third part,

[0029] And the remaining unmixed parts are independent.

[0030] In one embodiment, the two-part composition of the present invention comprises:

[0031] the first part (fibrinogen) mixed with the third part, and

[0032] The second part (thrombin) is independent.

[0033] In another embodiment, the present invention provides a three-part composition wherein the first part, the second part and the third part are independent.

[0034] Advantageously, the compositions of the present invention allow for accurate and precise printing of bone structures having defined, complex, and often irregular shapes. Thus, the compositions of the present invention address important unmet needs surrounding bone graft shape and size, which can be problematic for cadaveric and autologous bone grafts.

[0035] The components of the present invention are all natural or biocompatible materials and therefore present a low risk of toxicity. In addition, the individual portions of the composition of the present invention can be sterilized, thus reducing the risk of any associated infection after the bone construct is printed and implanted into the patient. Suitable, non-limiting sterilization methods include sterile filtration and radiation. In addition, the composition of the present invention can be printed in a sterile environment to produce a sterile bone construct.

[0036] Further advantageously, the composition of the present invention provides a bone construct with desirable structural properties and mechanical properties that can provide load-bearing support. In one embodiment, the composition of the present invention does not have any additional reinforcing material. For example, the multi-part composition of the present invention may not have any polymeric material or fibrous material, which adds additional strength and mechanical support to the bone construct after printing. Non-limiting examples of polymeric materials that increase strength and mechanical support may include poly (ethylene oxide)-poly (propylene oxide) copolymers. The fibrous material may be plant or animal derived. For example, the fibrous material may be derived from cotton, flax, hemp, jute, bamboo, recycled wood, waste paper, cellulose. In particular, the composition of the present invention may lack cellulose fibers.

[0037] Alternatively, in other embodiments, if necessary, the composition of the present invention may include additional reinforcing materials. For example, the multi-part composition of the present invention may include a polymeric material or a fibrous material, which adds additional strength and mechanical support to the bone construct after printing. Desirably, the polymeric material and the fibrous material are biocompatible. For example, the fibrous material may be of natural plant or animal origin. For example, the fibrous material may be selected from the group consisting of: cotton, flax, hemp, jute, bamboo, recycled wood, waste paper and cellulose. Non-limiting examples of polymeric materials that increase strength and mechanical support may include poly (ethylene oxide) -poly (propylene oxide) copolymers.

[0038] Further advantageously, the multi-part compositions of the present invention can provide a viable, non-toxic environment for bioactive materials such as cells, proteins and growth factors. Desirably, bioactive materials such as cells and proteins have osteoconductive properties, osteoinductive properties and / or osteogenic properties. For example, the bioactive materials can be immature cells that can differentiate into any cell type, bone morphogenetic proteins and combinations thereof. For example, the bioactive materials can include human mesenchymal stem cells (hMSC), bone morphogenetic proteins and combinations thereof.

[0039] As used herein, the terms osteoconduction, osteoinduction and osteogenesis are interrelated. In particular, osteogenesis is the process of bone formation. Osteoinduction is the process of inducing bone formation and is generally manifested as stimulating undifferentiated, immature cells to become active osteoblasts. Osteoconduction is a term used to describe a graft material that acts as a scaffold for new bone growth and facilitates new bone growth.

[0040] In one embodiment, the multi-part composition of the invention comprises hMSCs. The hMSCs may be present in the third part of the multi-part composition of the invention. Suitably, the hMSCs may be present in an amount of about 1*10 3 hMSCs to about 5*10 10 For example, hMSCs may be present at a concentration of about 1 to 10 hMSCs per mL of pharmaceutically acceptable hydrogel. 4 hMSCs to about 5*10 9 hMSCs, such as about 1*10 5 hMSCs to about 5*10 8 hMSCs, for example, about 1*10 5 hMSCs to about 5*10 7 In one embodiment, the hMSCs may be present at a concentration of about 1 to 10 hMSCs per mL of the pharmaceutically acceptable hydrogel. 6 hMSCs to about 9*10 6 hMSCs exist at concentrations between 1 and 2.

[0041] In other embodiments, the multi-part composition of the present invention may include a pharmacologically active agent and may be used as a depot or reservoir thereof. For example, the composition of the present invention may include a chemotherapeutic agent, an anti-tumor agent, an anti-inflammatory agent, an anti-infective agent, and combinations thereof.

[0042] Fibrinogen and thrombin

[0043] The fibrinogen used in the present invention can be obtained and purified from human plasma. Selectively, the fibrinogen used in the present invention can be recombinant, and obtains and purifies from a recombinant process. In one embodiment, the first portion of the multi-part composition of the present invention can include and be between about 5mg / mL to about 200mg / mL, for example from about 25mg / mL to about 175mg / mL, such as about 50mg / mL to about 150mg / mL, suitably from about 75mg / mL to about 125mg / mL The fibrinogen of the concentration. In one embodiment, the first portion of the multi-part composition of the present invention can include the fibrinogen from about 75mg / mL to about 100mg / mL. The first portion of the multi-part composition of the present invention can include the fibrinogen of about 80mg / mL.

[0044] Similarly, the thrombin used in the present invention can be obtained and purified from human plasma. Selectively, the thrombin used in the present invention can be recombinant, and obtains and purifies from a recombinant process. In one embodiment, the second portion of the multi-part compositions of the present invention can comprise and be between about 25IU / mL and 1500IU / mL, for example from about 50IU / mL to about 1250IU / mL, such as about 75IU / mL to about 1000IU / mL, suitably from about 100IU / mL to about 750IU / mL, for example from about 250IU / mL to about 750IU / mL, such as from about 400IU / mL to the thrombin of the concentration of about 600IU / mL. In one embodiment, the second portion of the multi-part compositions of the present invention can comprise the thrombin from about 450IU / mL to about 550IU / mL. The second portion of the multi-part compositions of the present invention can comprise the thrombin of about 500IU / mL.

[0045] The skilled person will appreciate that fibrinogen and thrombin are components in the human coagulation cascade. Thrombin acts on fibrinogen to produce polymer fibrin, which causes a gel type transition when the multi-part composition of the present invention is mixed. The conversion of fibrinogen (in the first part) to fibrin by the action of thrombin (in the second part) does not need to be 100% quantitative. The present invention also contemplates non-quantitative conversions, wherein the final bone construct may comprise a mixture of fibrinogen, fibrin and thrombin. In addition, when using the terms fibrinogen and thrombin, this specification includes within its scope derivatives of fibrinogen and thrombin, the derivatives of the fibrinogen and thrombin:

[0046] 1. does not substantially alter the interaction between the two proteins; or

[0047] 2. Do not substantially change the final fibrin product obtained.

[0048] Desirably, the first part of the multi-part composition of the present invention comprises fibrinogen formulated in a pharmaceutically acceptable vehicle comprising at least one amino acid. The amino acid can be selected from the group consisting of arginine, lysine, histidine, glutamic acid, aspartic acid, alanine, valine, leucine, isoleucine, and combinations thereof. For example, the amino acid can be selected from the group consisting of arginine, glutamic acid, isoleucine, and combinations thereof. In one embodiment, the first part of the multi-part composition of the present invention comprises fibrinogen formulated in a pharmaceutically acceptable vehicle comprising at least one amino acid and a citrate buffer. In other embodiments, the first part of the multi-part composition can additionally comprise a sodium salt, such as sodium chloride.

[0049] In yet another embodiment, the first part of the multi-part composition of the present invention comprises fibrinogen formulated in a pharmaceutically acceptable vehicle comprising at least one amino acid, a citrate buffer, and a sodium salt such as sodium chloride. In this embodiment, the amino acid can be selected from the group consisting of arginine, lysine, histidine, glutamic acid, aspartic acid, alanine, valine, leucine, isoleucine, and combinations thereof. For example, the amino acid can be selected from the group consisting of arginine, glutamic acid, isoleucine, and combinations thereof.

[0050] Preferably, the second part of the multi-part composition of the present invention comprises thrombin formulated in a pharmaceutically acceptable vehicle comprising dissolved calcium. The pharmaceutically acceptable vehicle may comprise a soluble calcium salt. For example, the calcium salt may be calcium chloride. In a further embodiment, the second part of the multi-part composition of the present invention may also comprise albumin and / or at least one amino acid. For example, the second part of the multi-part composition of the present invention may comprise a calcium salt and albumin (in addition to thrombin). For example, the second part of the multi-part composition of the present invention may comprise a calcium salt and at least one amino acid (in addition to thrombin). In one embodiment, the second part of the multi-part composition of the present invention may comprise a calcium salt, albumin and at least one amino acid (in addition to thrombin) having an uncharged side chain. In one embodiment, the amino acid is selected from the group consisting of glycine, alanine and a combination thereof. The calcium salt may be present in a concentration of about 0.01 mM to about 2.0 mM per IU of thrombin, for example about 0.01 mM to about 1.0 mM per IU of thrombin, such as about 0.01 mM to about 0.1 mM per IU of thrombin.

[0051] Hydrogel

[0052] Regarding the third part of the multi-part composition of the present invention, the pharmaceutically acceptable hydrogel can be selected from the group consisting of: hydrophilic polysaccharides, gelatin hydrogels and combinations thereof. Within this specification, reference to a constituent hydrophilic polymer of a hydrogel such as alginate, gelatin, etc. will be interpreted as reference to a hydrogel prepared using the hydrophilic polymer.

[0053] As used herein, the term hydrogel should be interpreted as a material having a three-dimensional (3D) network of hydrophilic polymers that can swell in water and retain large amounts of water while maintaining structure due to chemical or physical crosslinking of individual polymer chains. Hydrogels within the scope of the present invention may contain at least 10% w / w water, for example at least 20% w / w water, such as at least 30% w / w water. The hydrogel may contain greater than 40% w / w water; in some embodiments, the hydrogel may contain greater than 50% w / w water.

[0054] For example, the pharmaceutically acceptable hydrogel can be selected from the group consisting of: alginate, hyaluronic acid, gelatin, and combinations thereof. In one embodiment, the pharmaceutically acceptable hydrogel can be selected from the group consisting of: alginate, hyaluronic acid, and combinations thereof. In one embodiment, the pharmaceutically acceptable hydrogel is alginate.

[0055] As used herein, the term alginate refers to a naturally occurring anionic polymer typically obtained from natural sources such as seaweed or bacteria. The material is biocompatible, has low toxicity, and can be synthesized by adding divalent cations such as Ca 2+ Alginate is a block copolymer comprising a block of (1,4)-connected β-D-mannuronic acid (M) residues and α-L-guluronic acid (G) residues. Blocks include continuous G residues (e.g., GGGGGG), continuous M residues (e.g., MMMMMM) and alternating M residues and G residues (e.g., GMGMGM). Alginate extracted from different sources differs in M ​​content and G content and the length of each block. Without intending to limit the present invention by theory, it is believed that the G-block of alginate is considered to participate in intermolecular crosslinking to form a hydrogel.

[0056] Alginates within the scope of the present invention include simple chemical derivatives of (1,4)-linked β-D-mannuronic acid (M) units and α-L-guluronic acid (G) units, such as but not limited to ether derivatives, ester derivatives, carbonate derivatives and urethane derivatives.

[0057] Alginate within the scope of the present invention can have a molecular weight between about 10,000g / mol and about 500,000g / mol. For example, alginate within the scope of the present invention can have from about 40,000g / mol to about 400,000g / mol, such as about 50,000g / mol to about 300,000g / mol, suitably from about 60,000g / mol to about 250,000g / mol molecular weight. In one embodiment, alginate for the present invention can have a molecular weight of about 75,000g / mol to about 200,000g / mol.

[0058] Alginate that can be used for the present invention can have the molecular weight between about 10kDa and about 500kDa.For example, alginate within the scope of the present invention can have from about 40kDa to about 400kDa, such as about 50kDa to about 300kDa, suitably from about 60kDa to about 250kDa molecular weight.In one embodiment, alginate that is used for the present invention can have the molecular weight of about 75kDa to about 200kDa.

[0059] Alginates useful in the compositions of the invention may have a G / M ratio of ≤2.5, such as ≤2, for example ≤1.5, such as ≤1.0, suitably ≤0.5. In one embodiment, alginates useful in the compositions of the invention may have a G / M ratio of ≤1.

[0060] Alginates suitable for use in the compositions of the present invention may have a viscosity in 10 s as measured by a rotational viscometer at 25° C. and a pressure of 1 atmosphere. -1 The apparent viscosity of the shear rate is about 1 Pa·s to about 100 Pa·s, such as about 1 Pa·s to about 80 Pa·s, for example about 1 Pa·s to about 60 Pa·s, suitably about 3 Pa·s to about 40 Pa·s, such as about 3 Pa·s to about 30 Pa·s, for example about 4 Pa·s to about 30 Pa·s, suitably about 4 Pa·s to about 25 Pa·s.

[0061] Alginates suitable for use in the compositions of the present invention may have a molecular weight between 50,000 g / mol and about 300,000 g / mol, and a G / M ratio of ≤ 2. Alginates suitable for use in the compositions of the present invention may have a molecular weight between 50,000 g / mol and about 300,000 g / mol, and a viscosity within 10 s as measured by a rotational viscometer at 25° C. and a pressure of 1 atmosphere. -1 The apparent viscosity of the sample is from about 1 Pa·s to about 60 Pa·s at a shear rate of about 1 Pa·s.

[0062] In another embodiment, alginate suitable for use in the composition of the present invention may have a molecular weight between 75,000 g / mol and about 200,000 g / mol, and a G / M ratio of ≤ 1. Alternatively, alginate suitable for use in the composition of the present invention may have a molecular weight between 75,000 g / mol and about 200,000 g / mol, and a viscosity within 10 s as measured by a rotational viscometer at 25° C. and a pressure of 1 atmosphere. -1 The apparent viscosity of the sample is from about 1 Pa·s to about 60 Pa·s at a shear rate of about 1 Pa·s.

[0063] Alginates suitable for use in the compositions of the present invention may have a molecular weight between 50,000 g / mol and about 300,000 g / mol, a G / M ratio of ≤ 2, and a viscosity within 10 s as measured by a rotational viscometer at 25° C. and a pressure of 1 atmosphere. -1 The apparent viscosity of the sample is from about 1 Pa·s to about 60 Pa·s at a shear rate of about 1 Pa·s.

[0064] In another embodiment, alginate suitable for use in the compositions of the present invention may have a molecular weight between 75,000 g / mol and about 200,000 g / mol, a G / M ratio of ≤ 1, and a viscosity within 10 s as measured by a rotational viscometer at 25° C. and a pressure of 1 atmosphere. -1 The apparent viscosity of the sample is from about 1 Pa·s to about 60 Pa·s at a shear rate of about 1 Pa·s.

[0065] As used herein, the term hyaluronic acid refers to a glycosaminoglycan polysaccharide comprising a repeating disaccharide of β4-glucuronic acid-β3-N-acetylglucosamine. Hyaluronic acid polymers are commercially produced in large quantities by extracting the material from animal tissues or by recombinant expression in suitable organisms such as bacteria.

[0066] The hyaluronic acid used to prepare the hydrogel of the present invention can have a molecular weight of about 5 kDa to about 10,000 kDa, for example about 50 kDa to about 9000 kDa, such as about 100 kDa to about 8000 kDa, suitably about 500 kDa to about 7000 kDa, for example about 1000 kDa to about 5000 kDa, such as about 1000 kDa to about 4000 kDa, suitably about 1000 kDa to about 3000 kDa, for example about 1000 kDa to about 2500 kDa, such as about 1500 kDa to about 2500 kDa.

[0067] Hyaluronic acid suitable for use in the composition of the present invention may have a viscosity in 10 s as measured by a rotational viscometer at 25° C. and a pressure of 1 atmosphere. -1The apparent viscosity of the shear rate is about 1 Pa·s to about 100 Pa·s, such as about 1 Pa·s to about 80 Pa·s, for example about 1 Pa·s to about 60 Pa·s, suitably about 3 Pa·s to about 40 Pa·s, such as about 3 Pa·s to about 30 Pa·s, for example about 4 Pa·s to about 30 Pa·s, suitably about 4 Pa·s to about 25 Pa·s.

[0068] Hyaluronic acid suitable for use in the compositions of the present invention may have a molecular weight of about 1000 kDa to about 4000 kDa and a viscosity in 10 s as measured by a rotational viscometer at 25° C. and a pressure of 1 atmosphere. -1 For example, hyaluronic acid suitable for use in the composition of the present invention may have a molecular weight of about 1000 kDa to about 2500 kDa, and an apparent viscosity of about 1 Pa·s to about 60 Pa·s as measured by a rotational viscometer at 25° C. and a pressure of 1 atmosphere.

[0069] As used herein, the term gelatin refers to a mixture of peptides and proteins produced by thermal hydrolysis of collagen extracted from the skin, bones, tendons and white connective tissue of animals such as domestic cattle, chickens, pigs, fish and even some insects. The source of gelatin from animals is skin and bone, and the source of gelatin from plants is starch, alginate, pectin, agar and carrageenan. Gelatin is a heterogeneous mixture of high molecular weight polypeptides that can swell and absorb 5-10 times their weight of water to form a hydrogel. Gelatin is biodegradable and non-toxic.

[0070] Gelatin suitable for use in the composition of the present invention may have a viscosity in 10 s as measured by a rotational viscometer at 25° C. and a pressure of 1 atmosphere. -1 The apparent viscosity of the shear rate is about 1 Pa·s to about 100 Pa·s, such as about 1 Pa·s to about 80 Pa·s, for example about 1 Pa·s to about 60 Pa·s, suitably about 3 Pa·s to about 40 Pa·s, such as about 3 Pa·s to about 30 Pa·s, for example about 4 Pa·s to about 30 Pa·s, suitably about 4 Pa·s to about 25 Pa·s.

[0071] Biocompatible Inorganic Materials

[0072] Regarding the third part of the multi-part composition of the present invention, the biocompatible inorganic material can provide a source of both calcium atoms and phosphorus atoms. For example, the biocompatible inorganic material can be selected from the group consisting of: bioglass, tricalcium phosphate, single-phase hydroxyapatite, biphasic hydroxyapatite-tricalcium phosphate, natural bone powder and combinations thereof.

[0073] As used herein, the term bioglass refers to a sodium calcium phosphosilicate material.

[0074] In yet further embodiments, the biocompatible inorganic material may be selected from the group consisting of tricalcium phosphate, single-phase hydroxyapatite, biphasic hydroxyapatite-tricalcium phosphate, and combinations thereof.The tricalcium phosphate used in the composition of the present invention may be β-tricalcium phosphate.

[0075] For compatibility with 3D printing processes and equipment, preferably, the biocompatible inorganic material has an average particle size of less than about 200 μm. For example, the biocompatible inorganic material may have an average particle size of less than about 150 μm. Suitably, the biocompatible inorganic material may have an average particle size of less than about 100 μm. For example, the biocompatible inorganic material may have an average particle size between about 50 μm and about 200 μm.

[0076] The biocompatible inorganic material can be incorporated into the third part of the multi-part composition of the present invention at a concentration of between about 1 g to about 10 g of the biocompatible inorganic material per 1 mL of hydrogel. For example, about 2 g to about 8 g, such as about 2 g to about 6 g of the biocompatible inorganic material per 1 mL of hydrogel. In one embodiment, the biocompatible inorganic material can be incorporated into the third part of the multi-part composition of the present invention at a concentration of between about 3 g to about 6 g of the biocompatible inorganic material per 1 mL of hydrogel.

[0077] In some embodiments, the biocompatible inorganic material of the present invention can be hydrated with an albumin solution prior to mixing with a pharmaceutically acceptable hydrogel. The albumin can be from any suitable source such as human, bovine, or recombinant in origin. Preferably, the albumin is human albumin. The albumin solution can be saline-based. In certain embodiments, the albumin solution can contain between about 0.01% w / v to about 10% w / v albumin. For example, between about 0.1% w / v to about 5% w / v albumin, such as between about 1% w / v to about 3% w / v albumin.

[0078] In one embodiment, the preferred biocompatible inorganic material used in the composition of the present invention is β-tricalcium phosphate (β-TCP). β-TCP can have a rhombohedral lattice with an R-3c space group. β-TCP can be characterized by an X-ray powder diffraction pattern comprising the following ° 2θ (d values) when obtained using a Cu tube anode with K-alpha radiation: ) at angles of 17.0 (5.2), 21.9 (4.1), 25.8 (3.45), 27.8 (3.2), 29.65 (3.0), 31.0 (2.9), 32.45 (2.75), 34.4 (2.6), 46.9 (1.9), 48.0 (1.9), 48.4 (1.9) and 53.0 (1.7).

[0079] β-TCP may have a relative humidity of about 2.9 g / cm2 as determined by helium pycnometry. 3 and about 3.2g / cm 3 For example, β-TCP may have a density between about 2.9 g / cm 3 and about 3.15g / cm 3 For example, β-TCP may have a density between about 2.9 g / cm 3 and about 3.1g / cm 3 In one embodiment, β-TCP may have a density of about 2.95 g / cm 3 and about 3.15g / cm 3 In one embodiment, β-TCP may have a density of about 2.95 g / cm 3 and about 3.1g / cm 3 In one embodiment, β-TCP may have a density of about 3.0 g / cm 3 and about 3.1g / cm 3 The density between.

[0080] The β-TCP particles may have a d90 particle size distribution of no greater than about 180 μm. In one embodiment, the β-TCP particles may have a d90 particle size distribution of no greater than about 160 μm. In another embodiment, the β-TCP particles may have a d90 particle size distribution of no greater than about 140 μm.

[0081] In one embodiment, the β-TCP used in the composition of the present invention can be characterized by:

[0082] a d90 particle size distribution of no greater than about 180 μm, and

[0083] The average relative humidity was about 2.9 g / cm2 as determined by helium pycnometry. 3 and about 3.15g / cm 3 The density between.

[0084] In one embodiment, the β-TCP used in the composition of the present invention can be characterized by:

[0085] a d90 particle size distribution of no greater than about 160 μm, and

[0086] The average relative humidity was about 2.95 g / cm2 as determined by helium pycnometry. 3 and about 3.15g / cm 3 The density between.

[0087] In one embodiment, the β-TCP used in the composition of the present invention can be characterized by:

[0088] a d90 particle size distribution of no greater than about 160 μm, and

[0089] The average relative humidity was about 2.95 g / cm2 as determined by helium pycnometry. 3 and about 3.1g / cm 3 The density between.

[0090] In one embodiment, the β-TCP used in the composition of the present invention can be characterized by:

[0091] When using a Cu tube anode with K-alpha radiation, the following °2θ (d value X-ray powder diffraction pattern with unique peaks at ) at angles of 17.0 (5.2), 21.9 (4.1), 25.8 (3.45), 27.8 (3.2), 29.65 (3.0), 31.0 (2.9), 32.45 (2.75), 34.4 (2.6), 46.9 (1.9), 48.0 (1.9), 48.4 (1.9) and 53.0 (1.7).

[0092] The average relative humidity was about 2.95 g / cm2 as determined by helium pycnometry. 3 and about 3.15g / cm 3 The density between

[0093] A d90 particle size distribution of no greater than about 180 μm.

[0094] In one embodiment, the β-TCP used in the composition of the present invention can be characterized by:

[0095] When using a Cu tube anode with K-alpha radiation, the following °2θ (d value X-ray powder diffraction pattern with unique peaks at ) at angles of 17.0 (5.2), 21.9 (4.1), 25.8 (3.45), 27.8 (3.2), 29.65 (3.0), 31.0 (2.9), 32.45 (2.75), 34.4 (2.6), 46.9 (1.9), 48.0 (1.9), 48.4 (1.9) and 53.0 (1.7).

[0096] The average relative humidity was about 2.95 g / cm2 as determined by helium pycnometry. 3 and about 3.1g / cm 3 The density between

[0097] A d90 particle size distribution of no greater than about 160 μm.

[0098] Part III Composition

[0099] With regard to the third part of the composition of the present invention, i.e., the mixture comprising the pharmaceutically acceptable hydrogel and the biocompatible inorganic material, in some embodiments, the third part has an apparent viscosity selected from the group consisting of:

[0100] The viscosity of the sample was measured by a rotational viscometer at 25°C and a pressure of 1 atm in 0.1 s. -1 A shear rate of from about 30 Pa·s to about 300 Pa·s, and

[0101] The viscosity of the sample was measured by a rotational viscometer at 25 °C and a pressure of 1 atm in 1 s. -1 The shear rate is from about 1 Pa·s to about 75 Pa·s.

[0102] In further embodiments, the third part of the multi-part composition of the present invention may have an apparent viscosity selected from the group consisting of:

[0103] The viscosity of the sample was measured by a rotational viscometer at 25°C and a pressure of 1 atm in 0.1 s. -1 A shear rate of from about 30 Pa·s to about 250 Pa·s, and

[0104] The viscosity of the sample was measured by a rotational viscometer at 25 °C and a pressure of 1 atm in 1 s. -1 The shear rate is from about 5 Pa·s to about 75 Pa·s.

[0105] For example, the third part of the multi-part composition of the present invention may have an apparent viscosity selected from the group consisting of:

[0106] The viscosity of the sample was measured by a rotational viscometer at 25°C and a pressure of 1 atm in 0.1 s. -1A shear rate of from about 30 Pa·s to about 200 Pa·s, and

[0107] The viscosity of the sample was measured by a rotational viscometer at 25 °C and a pressure of 1 atm in 1 s. -1 The shear rate is from about 5 Pa·s to about 50 Pa·s.

[0108] Suitably, the third part of the multi-part composition of the invention may have an apparent viscosity selected from the group consisting of:

[0109] The viscosity of the sample was measured by a rotational viscometer at 25°C and a pressure of 1 atm in 0.1 s. -1 A shear rate of from about 50 Pa·s to about 200 Pa·s, and

[0110] The viscosity of the sample was measured by a rotational viscometer at 25 °C and a pressure of 1 atm in 1 s. -1 The shear rate is from about 10 Pa·s to about 45 Pa·s.

[0111] In one embodiment, the third part of the multi-part composition of the present invention may have an apparent viscosity selected from the group consisting of:

[0112] The viscosity of the sample was measured by a rotational viscometer at 25°C and a pressure of 1 atm in 0.1 s. -1 A shear rate of from about 100 Pa·s to about 300 Pa·s, and

[0113] The viscosity of the sample was measured by a rotational viscometer at 25 °C and a pressure of 1 atm in 1 s. -1 The shear rate is from about 1 Pa·s to about 50 Pa·s.

[0114] Non-limiting embodiments

[0115] In certain embodiments, the multi-part compositions of the present invention may comprise or consist essentially of:

[0116] i) a first part comprising fibrinogen in a pharmaceutically acceptable vehicle comprising at least one amino acid selected from the group consisting of arginine, lysine, histidine, glutamic acid, aspartic acid, alanine, valine, leucine, isoleucine, and combinations thereof;

[0117] ii) a second part comprising thrombin in a pharmaceutically acceptable carrier; and

[0118] iii) a third part comprising a pharmaceutically acceptable hydrogel mixed with at least one biocompatible inorganic material, the at least one inorganic material providing a source of at least one of calcium atoms and phosphorus atoms,

[0119] wherein the third part has an apparent viscosity selected from the group consisting of:

[0120] The viscosity of the sample was measured by a rotational viscometer at 25°C and a pressure of 1 atm in 0.1 s. -1 A shear rate of from about 1 Pa·s to about 300 Pa·s, and

[0121] The viscosity of the sample was measured by a rotational viscometer at 25 °C and a pressure of 1 atm in 1 s. -1 The shear rate is from about 1 Pa·s to about 100 Pa·s.

[0122] In certain embodiments, the multi-part compositions of the present invention may comprise or consist essentially of:

[0123] i) a first part comprising fibrinogen in a pharmaceutically acceptable vehicle comprising at least one amino acid selected from the group consisting of arginine, lysine, histidine, glutamic acid, aspartic acid, alanine, valine, leucine, isoleucine, and combinations thereof;

[0124] ii) a second part comprising thrombin in a pharmaceutically acceptable carrier; and

[0125] iii) a third part comprising alginate hydrogel mixed with at least one biocompatible inorganic material, the at least one inorganic material providing a source of at least one of calcium atoms and phosphorus atoms,

[0126] wherein the third part has an apparent viscosity selected from the group consisting of:

[0127] The viscosity of the sample was measured by a rotational viscometer at 25°C and a pressure of 1 atm in 0.1 s. -1 A shear rate of from about 50 Pa·s to about 250 Pa·s, and

[0128] The viscosity of the sample was measured by a rotational viscometer at 25 °C and a pressure of 1 atm in 1 s. -1 The shear rate is from about 2 Pa·s to about 50 Pa·s.

[0129] In certain embodiments, the multi-part compositions of the present invention may comprise or consist essentially of:

[0130] i) a first part comprising fibrinogen in a pharmaceutically acceptable vehicle comprising at least one amino acid selected from the group consisting of arginine, lysine, histidine, glutamic acid, aspartic acid, alanine, valine, leucine, isoleucine, and combinations thereof;

[0131] ii) a second part comprising thrombin in a pharmaceutically acceptable carrier; and

[0132] iii) a third part comprising a pharmaceutically acceptable hydrogel selected from the group consisting of alginate, hyaluronic acid, gelatin, and combinations thereof, mixed with at least one biocompatible inorganic material, the at least one inorganic material providing a source of at least one of calcium atoms and phosphorus atoms,

[0133] wherein the third part has an apparent viscosity selected from the group consisting of:

[0134] The viscosity of the sample was measured by a rotational viscometer at 25°C and a pressure of 1 atm in 0.1 s. -1 A shear rate of from about 1 Pa·s to about 300 Pa·s, and

[0135] The viscosity of the sample was measured by a rotational viscometer at 25 °C and a pressure of 1 atm in 1 s. -1 The shear rate is from about 1 Pa·s to about 100 Pa·s.

[0136] In certain embodiments, the multi-part compositions of the present invention may comprise or consist essentially of:

[0137] i) a first part comprising fibrinogen in a pharmaceutically acceptable vehicle comprising at least one amino acid selected from the group consisting of arginine, lysine, histidine, glutamic acid, aspartic acid, alanine, valine, leucine, isoleucine, and combinations thereof;

[0138] ii) a second part comprising thrombin in a pharmaceutically acceptable carrier; and

[0139] iii) a third part, the third part comprising a pharmaceutically acceptable hydrogel mixed with at least one biocompatible inorganic material, the pharmaceutically acceptable hydrogel selected from the group consisting of alginate, hyaluronic acid, gelatin and combinations thereof, the biocompatible inorganic material selected from the group consisting of bioglass, tricalcium phosphate, monophasic hydroxyapatite, biphasic hydroxyapatite-tricalcium phosphate, natural bone powder and combinations thereof,

[0140] wherein the third part has an apparent viscosity selected from the group consisting of:

[0141] The viscosity of the sample was measured by a rotational viscometer at 25°C and a pressure of 1 atm in 0.1 s. -1 A shear rate of from about 1 Pa·s to about 300 Pa·s, and

[0142] The viscosity of the sample was measured by a rotational viscometer at 25 °C and a pressure of 1 atm in 1 s. -1 The shear rate is from about 1 Pa·s to about 100 Pa·s.

[0143] In certain embodiments, the multi-part compositions of the present invention may comprise or consist essentially of:

[0144] i) a first part comprising fibrinogen in a pharmaceutically acceptable vehicle comprising at least one amino acid selected from the group consisting of arginine, lysine, histidine, glutamic acid, aspartic acid, alanine, valine, leucine, isoleucine, and combinations thereof;

[0145] ii) a second part comprising thrombin in a pharmaceutically acceptable carrier comprising dissolved calcium, and an additional excipient selected from the group consisting of albumin, amino acids, and combinations thereof; and

[0146] iii) a third part comprising a pharmaceutically acceptable hydrogel mixed with at least one biocompatible inorganic material selected from the group consisting of bioglass, tricalcium phosphate, monophasic hydroxyapatite, biphasic hydroxyapatite-tricalcium phosphate, natural bone powder, and combinations thereof,

[0147] wherein the third part has an apparent viscosity selected from the group consisting of:

[0148] The viscosity of the sample was measured by a rotational viscometer at 25°C and a pressure of 1 atm in 0.1 s. -1 A shear rate of from about 1 Pa·s to about 300 Pa·s, and

[0149] The viscosity of the sample was measured by a rotational viscometer at 25 °C and a pressure of 1 atm in 1 s. -1 The shear rate is from about 1 Pa·s to about 100 Pa·s.

[0150] In certain embodiments, the multi-part compositions of the present invention may comprise or consist essentially of:

[0151] i) a first part comprising fibrinogen in a pharmaceutically acceptable vehicle;

[0152] ii) a second part comprising thrombin in a pharmaceutically acceptable carrier comprising dissolved calcium, and an additional excipient selected from the group consisting of albumin, amino acids, and combinations thereof; and

[0153] iii) a third part comprising a pharmaceutically acceptable hydrogel mixed with at least one biocompatible inorganic material, the at least one inorganic material providing a source of at least one of calcium atoms and phosphorus atoms,

[0154] wherein the third part has an apparent viscosity selected from the group consisting of:

[0155] The viscosity of the sample was measured by a rotational viscometer at 25°C and a pressure of 1 atm in 0.1 s. -1 A shear rate of from about 1 Pa·s to about 300 Pa·s, and

[0156] The viscosity of the sample was measured by a rotational viscometer at 25 °C and a pressure of 1 atm in 1 s. -1 The shear rate is from about 1 Pa·s to about 100 Pa·s.

[0157] In certain embodiments, the multi-part compositions of the present invention may comprise or consist essentially of:

[0158] i) a first part comprising fibrinogen in a pharmaceutically acceptable vehicle;

[0159] ii) a second part comprising thrombin in a pharmaceutically acceptable carrier comprising dissolved calcium, and an additional excipient selected from the group consisting of albumin, amino acids, and combinations thereof; and

[0160] iii) a third part comprising a pharmaceutically acceptable hydrogel selected from the group consisting of alginate, hyaluronic acid, gelatin, and combinations thereof, mixed with at least one biocompatible inorganic material, the at least one inorganic material providing a source of at least one of calcium atoms and phosphorus atoms,

[0161] wherein the third part has an apparent viscosity selected from the group consisting of:

[0162] The viscosity of the sample was measured by a rotational viscometer at 25°C and a pressure of 1 atm in 0.1 s. -1 A shear rate of from about 1 Pa·s to about 300 Pa·s, and

[0163] The viscosity of the sample was measured by a rotational viscometer at 25 °C and a pressure of 1 atm in 1 s. -1The shear rate is from about 1 Pa·s to about 100 Pa·s.

[0164] In certain embodiments, the multi-part compositions of the present invention may comprise or consist essentially of:

[0165] i) a first part comprising fibrinogen in a pharmaceutically acceptable vehicle;

[0166] ii) a second part comprising thrombin in a pharmaceutically acceptable carrier comprising dissolved calcium, and an additional excipient selected from the group consisting of albumin, amino acids, and combinations thereof; and

[0167] iii) a third part, the third part comprising a pharmaceutically acceptable hydrogel mixed with at least one biocompatible inorganic material, the pharmaceutically acceptable hydrogel selected from the group consisting of alginate, hyaluronic acid, gelatin and combinations thereof, the biocompatible inorganic material selected from the group consisting of bioglass, tricalcium phosphate, monophasic hydroxyapatite, biphasic hydroxyapatite-tricalcium phosphate, natural bone powder and combinations thereof,

[0168] wherein the third part has an apparent viscosity selected from the group consisting of:

[0169] The viscosity of the sample was measured by a rotational viscometer at 25°C and a pressure of 1 atm in 0.1 s. -1 A shear rate of from about 1 Pa·s to about 300 Pa·s, and

[0170] The viscosity of the sample was measured by a rotational viscometer at 25 °C and a pressure of 1 atm in 1 s. -1 The shear rate is from about 1 Pa·s to about 100 Pa·s.

[0171] In certain embodiments, the multi-part compositions of the present invention may comprise or consist essentially of:

[0172] i) a first part comprising fibrinogen in a pharmaceutically acceptable vehicle;

[0173] ii) a second part comprising thrombin in a pharmaceutically acceptable carrier comprising dissolved calcium, and an additional excipient selected from the group consisting of albumin, amino acids, and combinations thereof; and

[0174] iii) a third part comprising a pharmaceutically acceptable hydrogel mixed with at least one biocompatible inorganic material, the at least one inorganic material providing a source of at least one of calcium atoms and phosphorus atoms,

[0175] wherein the third part has an apparent viscosity selected from the group consisting of:

[0176] The viscosity of the sample was measured by a rotational viscometer at 25°C and a pressure of 1 atm in 0.1 s. -1 A shear rate of from about 50 Pa·s to about 250 Pa·s, and

[0177] The viscosity of the sample was measured by a rotational viscometer at 25 °C and a pressure of 1 atm in 1 s. -1 The shear rate is from about 2 Pa·s to about 50 Pa·s.

[0178] In certain embodiments, the multi-part compositions of the present invention may comprise or consist essentially of:

[0179] i) a first part comprising fibrinogen in a pharmaceutically acceptable vehicle comprising at least one amino acid selected from the group consisting of arginine, lysine, histidine, glutamic acid, aspartic acid, alanine, valine, leucine, isoleucine, and combinations thereof;

[0180] ii) a second part comprising thrombin in a pharmaceutically acceptable carrier comprising dissolved calcium, and an additional excipient selected from the group consisting of albumin, amino acids, and combinations thereof; and

[0181] iii) a third part comprising a pharmaceutically acceptable hydrogel mixed with at least one biocompatible inorganic material, the at least one inorganic material providing a source of at least one of calcium atoms and phosphorus atoms,

[0182] wherein the third part has an apparent viscosity selected from the group consisting of:

[0183] The viscosity of the sample was measured by a rotational viscometer at 25°C and a pressure of 1 atm in 0.1 s. -1 A shear rate of from about 1 Pa·s to about 300 Pa·s, and

[0184] The viscosity of the sample was measured by a rotational viscometer at 25 °C and a pressure of 1 atm in 1 s. -1 The shear rate is from about 1 Pa·s to about 100 Pa·s.

[0185] In certain embodiments, the multi-part compositions of the present invention may comprise or consist essentially of:

[0186] i) a first part comprising fibrinogen in a pharmaceutically acceptable vehicle comprising at least one amino acid selected from the group consisting of arginine, lysine, histidine, glutamic acid, aspartic acid, alanine, valine, leucine, isoleucine, and combinations thereof;

[0187] ii) a second part comprising thrombin in a pharmaceutically acceptable carrier comprising dissolved calcium, and an additional excipient selected from the group consisting of albumin, amino acids, and combinations thereof; and

[0188] iii) a third part comprising a pharmaceutically acceptable hydrogel mixed with at least one biocompatible inorganic material, the at least one inorganic material providing a source of at least one of calcium atoms and phosphorus atoms,

[0189] wherein the third part has an apparent viscosity selected from the group consisting of:

[0190] The viscosity of the sample was measured by a rotational viscometer at 25°C and a pressure of 1 atm in 0.1 s. -1 A shear rate of from about 50 Pa·s to about 250 Pa·s, and

[0191] The viscosity of the sample was measured by a rotational viscometer at 25 °C and a pressure of 1 atm in 1 s. -1 The shear rate is from about 2 Pa·s to about 50 Pa·s.

[0192] In certain embodiments, the multi-part compositions of the present invention may comprise or consist essentially of:

[0193] i) a first part comprising fibrinogen in a pharmaceutically acceptable vehicle comprising at least one amino acid selected from the group consisting of arginine, lysine, histidine, glutamic acid, aspartic acid, alanine, valine, leucine, isoleucine, and combinations thereof;

[0194] ii) a second part comprising thrombin in a pharmaceutically acceptable carrier comprising dissolved calcium, and an additional excipient selected from the group consisting of albumin, amino acids, and combinations thereof; and

[0195] iii) a third part comprising a pharmaceutically acceptable hydrogel selected from the group consisting of alginate, hyaluronic acid, gelatin, and combinations thereof, mixed with at least one biocompatible inorganic material, the at least one inorganic material providing a source of at least one of calcium atoms and phosphorus atoms,

[0196] wherein the third part has an apparent viscosity selected from the group consisting of:

[0197] The viscosity of the sample was measured by a rotational viscometer at 25°C and a pressure of 1 atm in 0.1 s. -1 A shear rate of from about 1 Pa·s to about 300 Pa·s, and

[0198] The viscosity of the sample was measured by a rotational viscometer at 25 °C and a pressure of 1 atm in 1 s. -1 The shear rate is from about 1 Pa·s to about 100 Pa·s.

[0199] In certain embodiments, the multi-part compositions of the present invention may comprise or consist essentially of:

[0200] i) a first part comprising fibrinogen in a pharmaceutically acceptable vehicle comprising at least one amino acid selected from the group consisting of arginine, lysine, histidine, glutamic acid, aspartic acid, alanine, valine, leucine, isoleucine, and combinations thereof;

[0201] ii) a second part comprising thrombin in a pharmaceutically acceptable carrier comprising dissolved calcium, and an additional excipient selected from the group consisting of albumin, amino acids, and combinations thereof; and

[0202] iii) a third part, the third part comprising a pharmaceutically acceptable hydrogel mixed with at least one biocompatible inorganic material, the pharmaceutically acceptable hydrogel selected from the group consisting of alginate, hyaluronic acid, gelatin and combinations thereof, the biocompatible inorganic material selected from the group consisting of bioglass, tricalcium phosphate, monophasic hydroxyapatite, biphasic hydroxyapatite-tricalcium phosphate, natural bone powder and combinations thereof,

[0203] wherein the third part has an apparent viscosity selected from the group consisting of:

[0204] The viscosity of the sample was measured by a rotational viscometer at 25°C and a pressure of 1 atm in 0.1 s. -1 A shear rate of from about 1 Pa·s to about 300 Pa·s, and

[0205] The viscosity of the sample was measured by a rotational viscometer at 25 °C and a pressure of 1 atm in 1 s. -1 The shear rate is from about 1 Pa·s to about 100 Pa·s.

[0206] In certain embodiments, the multi-part compositions of the present invention may comprise or consist essentially of:

[0207] i) a first part comprising fibrinogen in a pharmaceutically acceptable vehicle comprising at least one amino acid selected from the group consisting of arginine, lysine, histidine, glutamic acid, aspartic acid, alanine, valine, leucine, isoleucine, and combinations thereof;

[0208] ii) a second part comprising thrombin in a pharmaceutically acceptable carrier comprising dissolved calcium, and an additional excipient selected from the group consisting of albumin, amino acids, and combinations thereof; and

[0209] iii) a third part comprising a pharmaceutically acceptable hydrogel selected from the group consisting of alginate, hyaluronic acid, gelatin, and combinations thereof, mixed with at least one biocompatible inorganic material, the at least one inorganic material providing a source of at least one of calcium atoms and phosphorus atoms,

[0210] wherein the third part has an apparent viscosity selected from the group consisting of:

[0211] The viscosity of the sample was measured by a rotational viscometer at 25°C and a pressure of 1 atm in 0.1 s. -1 A shear rate of from about 50 Pa·s to about 250 Pa·s, and

[0212] The viscosity of the sample was measured by a rotational viscometer at 25 °C and a pressure of 1 atm in 1 s. -1 The shear rate is from about 2 Pa·s to about 50 Pa·s.

[0213] In certain embodiments, the multi-part compositions of the present invention may comprise or consist essentially of:

[0214] i) a first part comprising fibrinogen in a pharmaceutically acceptable vehicle comprising at least one amino acid selected from the group consisting of arginine, lysine, histidine, glutamic acid, aspartic acid, alanine, valine, leucine, isoleucine, and combinations thereof;

[0215] ii) a second part comprising thrombin in a pharmaceutically acceptable carrier comprising dissolved calcium, and an additional excipient selected from the group consisting of albumin, amino acids, and combinations thereof; and

[0216] iii) a third part, the third part comprising a pharmaceutically acceptable hydrogel mixed with at least one biocompatible inorganic material, the pharmaceutically acceptable hydrogel selected from the group consisting of alginate, hyaluronic acid, gelatin and combinations thereof, the biocompatible inorganic material selected from the group consisting of bioglass, tricalcium phosphate, monophasic hydroxyapatite, biphasic hydroxyapatite-tricalcium phosphate, natural bone powder and combinations thereof,

[0217] wherein the third part has an apparent viscosity selected from the group consisting of:

[0218] The viscosity of the sample was measured by a rotational viscometer at 25°C and a pressure of 1 atm in 0.1 s. -1 A shear rate of from about 50 Pa·s to about 250 Pa·s, and

[0219] The viscosity of the sample was measured by a rotational viscometer at 25 °C and a pressure of 1 atm in 1 s. -1 The shear rate is from about 2 Pa·s to about 50 Pa·s.

[0220] In certain embodiments, the multi-part compositions of the present invention may comprise or consist essentially of:

[0221] i) a first part comprising fibrinogen in a pharmaceutically acceptable vehicle;

[0222] ii) a second part comprising thrombin in a pharmaceutically acceptable carrier; and

[0223] iii) a third part comprising a pharmaceutically acceptable hydrogel selected from the group consisting of alginate, hyaluronic acid, gelatin, and combinations thereof, mixed with at least one biocompatible inorganic material, the at least one inorganic material providing a source of at least one of calcium atoms and phosphorus atoms,

[0224] wherein the third part has an apparent viscosity selected from the group consisting of:

[0225] The viscosity of the sample was measured by a rotational viscometer at 25°C and a pressure of 1 atm in 0.1 s. -1 A shear rate of from about 50 Pa·s to about 250 Pa·s, and

[0226] The viscosity of the sample was measured by a rotational viscometer at 25 °C and a pressure of 1 atm in 1 s. -1 The shear rate is from about 2 Pa·s to about 50 Pa·s.

[0227] In certain embodiments, the multi-part compositions of the present invention may comprise or consist essentially of:

[0228] i) a first part comprising fibrinogen in a pharmaceutically acceptable vehicle;

[0229] ii) a second part comprising thrombin in a pharmaceutically acceptable carrier; and

[0230] iii) a third part, the third part comprising a pharmaceutically acceptable hydrogel mixed with at least one biocompatible inorganic material, the pharmaceutically acceptable hydrogel selected from the group consisting of alginate, hyaluronic acid, gelatin and combinations thereof, the biocompatible inorganic material selected from the group consisting of bioglass, tricalcium phosphate, monophasic hydroxyapatite, biphasic hydroxyapatite-tricalcium phosphate, natural bone powder and combinations thereof,

[0231] wherein the third part has an apparent viscosity selected from the group consisting of:

[0232] The viscosity of the sample was measured by a rotational viscometer at 25°C and a pressure of 1 atm in 0.1 s. -1 A shear rate of from about 50 Pa·s to about 250 Pa·s, and

[0233] The viscosity of the sample was measured by a rotational viscometer at 25 °C and a pressure of 1 atm in 1 s. -1 The shear rate is from about 2 Pa·s to about 50 Pa·s.

[0234] In all of the foregoing embodiments, alginates suitable for use in the compositions of the present invention may have a viscosity in 10 s as measured by a rotational viscometer at 25° C. and a pressure of 1 atmosphere. -1 An apparent viscosity of about 1 Pa·s to about 100 Pa·s, such as about 1 Pa·s to about 80 Pa·s, for example about 1 Pa·s to about 60 Pa·s, suitably about 3 Pa·s to about 40 Pa·s, such as about 3 Pa·s to about 30 Pa·s, for example about 4 Pa·s to about 30 Pa·s, suitably about 4 Pa·s to about 25 Pa·s at a shear rate. Alginates suitable for use in the compositions of the present invention may have a molecular weight between 50,000 g / mol and about 300,000 g / mol, and a G / M ratio of ≤2. Alginates suitable for use in the compositions of the present invention may have a molecular weight between 50,000 g / mol and about 300,000 g / mol, and a G / M ratio of ≤2 at 10 s. -1 The apparent viscosity of the sample is from about 1 Pa·s to about 60 Pa·s at a shear rate of about 1 Pa·s.

[0235] In all of the foregoing embodiments, alginates suitable for use in the compositions of the present invention may have a molecular weight between 75,000 g / mol and about 200,000 g / mol, and a G / M ratio of ≤ 1. Alternatively, alginates suitable for use in the compositions of the present invention may have a molecular weight between 75,000 g / mol and about 200,000 g / mol, and a viscosity within 10 s as measured by a rotational viscometer at 25° C. and a pressure of 1 atmosphere. -1 The apparent viscosity of the alginates suitable for use in the compositions of the present invention may be between 50,000 g / mol and about 300,000 g / mol, a G / M ratio of ≤ 2, and a viscosity of 10 s at a shear rate of about 1 Pa·s to about 60 Pa·s. The alginates suitable for use in the compositions of the present invention may have a molecular weight between 50,000 g / mol and about 300,000 g / mol, a G / M ratio of ≤ 2, and a viscosity of 10 s at 25° C. and a pressure of 1 atmosphere as measured by a rotational viscometer. -1 In another embodiment, the alginate suitable for use in the composition of the present invention may have a molecular weight between 75,000 g / mol and about 200,000 g / mol, a G / M ratio of ≤1, and an apparent viscosity of about 1 Pa·s to about 60 Pa·s at a shear rate of 10 s. -1 The apparent viscosity of the sample is from about 1 Pa·s to about 60 Pa·s at a shear rate of about 1 Pa·s.

[0236] Bone composite material of the present invention

[0237] In a further aspect, the present invention provides a bone composite material obtainable from the multi-part composition of the present invention.

[0238] In certain embodiments, the biocompatible inorganic material is in a concentration between about 5% w / w and about 60% w / w of the bone composite material of the present invention. For example, in a concentration between about 15% w / w and about 60% w / w, such as in a concentration between about 25% w / w and about 60% w / w, suitably in a concentration between about 35% w / w and about 60% w / w. In other embodiments, the biocompatible inorganic material is in a concentration selected from the group consisting of about 5% w / w to about 50% w / w, about 5% w / w to about 40% w / w, about 5% w / w to about 30% w / w, and about 10% w / w to about 25% w / w.

[0239] Method of the present invention

[0240] In yet another aspect, the present invention provides a method for preparing a bone composite material, the method comprising the steps of:

[0241] Providing a three-dimensional printing device having a multi-part composition of the present invention;

[0242] printing the bone composite material according to the determined design; and

[0243] The synthetic bone composite material is optionally incubated.

[0244] Naturally, due to the multi-part nature of the composition of the invention, a variety of different arrangements of the order in which the different parts are printed are possible and within the scope of the method of the invention. For example, the method of the invention for preparing a bone composite material may comprise printing with a three-dimensional printing device;

[0245] i) a first layer comprising fibrinogen or thrombin;

[0246] ii) a second layer comprising fibrinogen or thrombin;

[0247] wherein if the first layer comprises fibrinogen, the second layer comprises thrombin, and vice versa,

[0248] iii) repeating step i) and step ii) n times, wherein n ≥ 1, to produce an alternating layered structure;

[0249] iv) printing a third part comprising a hydrogel and a biocompatible inorganic material on top of the alternating layered structure of step iii); and

[0250] v) optionally repeating steps i) to iv) as needed to provide a bone composite material.

[0251] Referring to the method of the present invention, in one embodiment, fibrinogen is printed first and thrombin is printed second as a layer on top of the fibrinogen.

[0252] For example, referring to the embodiments of the methods of the present invention outlined previously,

[0253] The first layer may have a defined volume of X,

[0254] The second layer may have a bounded volume of Y,

[0255] Step i) and step ii) may be repeated n times, where n≥1, to produce an alternating layered structure having a volume nX+nY; and

[0256] A third part including a hydrogel and a biocompatible inorganic material is printed on top of the alternating layered structure, the third part having a volume Z.

[0257] In certain embodiments, volume X and volume Y are the same, such that volume nX=nY.

[0258] For example, in certain embodiments, the ratio of Z to nX to nY (Z:nX:nY) is about 0.60-1.0:1:1. Suitably, the ratio of Z to nX to nY (Z:nX:nY) is about 0.80-1.0:1:1. In some embodiments, the ratio of Z to nX to nY (Z:nX:nY) is about 0.90-0.99:1:1. For example, the ratio of Z to nX to nY (Z:nX:nY) can be about 0.95:1:1. In yet another embodiment, the three-dimensional printer can print the first part, the second part, and the third part of the multi-part composition of equal volume, so that the ratio of Z to nX to nY (Z:nX:nY) is about 1:1:1.

[0259] In other embodiments, the ratio of Z to nX to nY (Z:nX:nY) is about 1-5:1:1. Suitably, the ratio of Z to nX to nY (Z:nX:nY) is about 2-4:1:1. In some embodiments, the ratio of Z to nX to nY (Z:nX:nY) is about 3-4:1:1. For example, the ratio of Z to nX to nY (Z:nX:nY) can be about 4:1:1.

[0260] In some embodiments, 2≤n≤10.

[0261] In other embodiments of the method for preparing a bone composite material of the present invention, a three-dimensional printing device can print;

[0262] i) a first layer comprising the third part of the multi-part composition of the invention (i.e., the hydrogel and the biocompatible inorganic material);

[0263] ii) a second layer comprising fibrinogen or thrombin;

[0264] iii) a third layer comprising fibrinogen or thrombin;

[0265] wherein if the second layer comprises fibrinogen, the third layer comprises thrombin, and vice versa, and

[0266] iv) repeating step ii) and step iii) sequentially n times, wherein n≥1, to produce an alternating layered structure of fibrinogen and thrombin; and

[0267] v) optionally repeating steps i) to iv) as needed to provide a bone composite material.

[0268] For example, referring to the embodiments of the methods of the present invention outlined previously,

[0269] The first layer may have a defined volume of A,

[0270] The second layer may have a defined volume of B,

[0271] The third layer may have a defined volume of C,

[0272] Therein steps ii) and iii) can be repeated n times, where n≥1, to produce a layered structure having a volume A+nB+nC.

[0273] In certain embodiments, volume B and volume C are the same, such that volume nB=nC.

[0274] For example, in certain embodiments, the ratio of A to nB to nC (A:nB:nC) is about 1-5:1:1. Suitably, the ratio of A to nB to nC (A:nB:nC) is about 2-4:1:1. In some embodiments, the ratio of A to nB to nC (A:nB:nC) is about 3-4:1:1. For example, the ratio of A to nB to nC (A:nB:nC) can be about 4:1:1.

[0275] For example, in certain embodiments, the ratio of A to nB to nC (A:nB:nC) is about 0.60-1.0:1:1. Suitably, the ratio of A to nB to nC (A:nB:nC) is about 0.80-1.0:1:1. In some embodiments, the ratio of A to nB to nC (A:nB:nC) is about 0.90-0.99:1:1. For example, the ratio of A to nB to nC (A:nB:nC) can be about 0.95:1:1. In other embodiments, the three-dimensional printer can print the first part, the second part, and the third part of the multi-part composition of equal volume, so that the ratio of A to nB to nC (A:nB:nC) is about 1:1:1.

[0276] In some embodiments, 2≤n≤10.

[0277] For the two-part composition of the present invention, the method of the present invention may include a three-dimensional printing device:

[0278] i) printing a first part of the composition comprising fibrinogen or thrombin;

[0279] ii) repeating step i) n times, wherein n ≥ 1, to produce a layered structure comprising only one of fibrinogen or thrombin;

[0280] iii) printing a second part on top of the layered structure produced in step ii), the second part comprising the other of fibrinogen or thrombin mixed with a pharmaceutically acceptable hydrogel and a biocompatible inorganic material;

[0281] iv) optionally repeating steps i) to iii) as needed to provide a bone composite material.

[0282] Alternatively, for the two-part composition of the invention, the method of the invention may include a three-dimensional printing device:

[0283] i) printing a second part comprising one of fibrinogen or thrombin mixed with a pharmaceutically acceptable hydrogel and a biocompatible inorganic material;

[0284] ii) printing a first portion comprising the other of fibrinogen or thrombin on top of the portion printed in step i);

[0285] iii) repeating step ii) n times, wherein n ≥ 1, to produce a layered structure;

[0286] iv) optionally repeating steps i) to iii) as needed to provide a bone composite material.

[0287] With regard to embodiments involving printing of a two-part composition of the invention, the ratio of the volume of the printed first part to the volume of the printed second part (Part 1: Part 2) may be about 1: 1-8, for example about 1: 6, such as about 1: 4, suitably about 1: 2, for example about 1: 1. In certain embodiments, the ratio of the volume of the printed first part to the volume of the printed second part is 1: 2.

[0288] In a further aspect, the present invention provides a bone composite material obtainable by the method of the present invention.

[0289] Other aspects of the invention

[0290] In yet another aspect, the present invention provides a method of treating a bone injury or bone defect in a patient in need thereof, the method comprising the step of implanting the bone composite material of the present invention into a patient in need thereof.

[0291] A further aspect of the invention relates to the use of the bone composite material of the invention in the treatment of bone injuries or bone defects.The bone composite material of the invention may have particular use in the treatment of trauma and / or cancer patients.

[0292] In yet another aspect, the present invention provides a kit comprising:

[0293] 3D printers, and

[0294] Multi-part compositions of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0296] Further features and advantages of the present invention will become more apparent from the accompanying drawings, in which:

[0297] Figure 1The figure shows a series of printing tests for a three-part composition of the present invention. Structures with defined geometries were printed and the results for the different compositions are outlined in the grid array shown;

[0298] Figure 2 The figure shows a graph of hMSC cell proliferation in the composition of the present invention as measured by ATP cell proliferation assay (Promega);

[0299] Figure 3 Depicting a graph of hMSC cell viability in a composition of the present invention as measured using a Live / Dead Cell Viability Assay (Life Technologies);

[0300] Figure 4 shows the results of an hMSC osteoblast differentiation assay for a composition of the present invention; and

[0301] Figure 5 The figure shows a series of printing tests for a two-part composition of the invention.

[0302] Detailed description of the present invention

[0303] It should be readily apparent to those of ordinary skill in the art that the embodiments disclosed herein below represent only typical embodiments, and that other arrangements and methods capable of reproducing the present invention are possible and encompassed by the present invention.

[0304] Example 1

[0305] Objects having defined geometries are printed using the three-part composition of the present invention.

[0306] Advantageously, the compositions of the present invention allow accurate and precise printing of structures having defined and irregular shapes. A series of tests illustrating the performance of many of the multi-part compositions of the present invention are summarized below. The compositions are loaded into a 3D printing device and a computer design drawing provides the shape of interest. The operation of the 3D printing device is within the normal skills and capabilities of one of ordinary skill in the art.

[0307] The components of the three-part composition of the present invention are summarized below. Figure 1 All bone constructs shown in the figures comprise the same fibrinogen component (first part) and thrombin component (second part), which are qualitatively summarized in Table 1 below.

[0308] Fibrinogen fraction Thrombin components Human fibrinogen (80 mg / mL) Human thrombin (500 IU / mL) Sodium citrate dihydrate Calcium chloride Sodium chloride Human Albumin Arginine Sodium chloride Isoleucine Glycine Monosodium Glutamate Water for injection Water for injection

[0309] Table 1

[0310] The surgical sealant product VERASEAL, manufactured and sold by Grifols under marketing authorization number EU / 1 / 17 / 1239 / 001-004, was the source of the fibrinogen component and thrombin component used in all experiments outlined in Examples 1 to 5. This product is also sold in the United States by Grifols under Biologics License Number (BLN) 125640 as FIBRIN SEALANT.

[0311] The hydrogel compositions were then prepared using standard methods at the concentrations outlined in Table 2. For example, 5 g of sodium alginate [(75-200 kDa, G / M ratio ≤ 1, viscosity 20-200 mPa*s (PRONOVA UP LVM, DuPont)] was weighed out in a laminar flow cabinet. MilliQ water (100 mL) was heated to 50° C. and the sodium alginate was added with mixing in a laminar flow cabinet. The water was kept at a constant temperature of 50° C. until the sodium alginate powder was no longer visible (approximately 1 hour). The resulting solution was covered to maintain sterility and cooled to approximately 6° C. to accelerate the gelation process.

[0312] HYALUBRIX is used directly from the syringe.

[0313]

[0314]

[0315] Table 2

[0316] Table 3 summarizes the ingredients of the third part of the multi-part composition of the present invention and their concentrations. The weights of biocompatible inorganic materials (bioglass, TCP, etc.) indicated in Table 3 were hydrated with 8 mL of albumin hydration solution and manually mixed before incorporating them into the hydrogel. The albumin hydration solution consisted of human serum albumin at a concentration of 20 mg / mL in a saline vehicle. Both saline and human serum albumin [ALBUTEIN] were obtained from Grifols.

[0317] The resulting suspension was centrifuged at 140 g for 2 minutes. Excess hydration solution was removed using a micropipette. The hydrated biocompatible inorganic material was combined with the hydrogels outlined in Table 2 using manual mixing. The resulting composition is outlined in Table 3. The physical properties of the biocompatible inorganic material are listed below for reference:

[0318]

[0319] The #1-9 numbers used in Table 3 correspond directly to Figure 1 Number of bone constructs shown in the figure.

[0320]

[0321]

[0322] *=75%hydroxyapatite(HA):25%β-TCP

[0323] Table 3

[0324] The 3D printing processes for printing the compositions outlined in Tables 1 and 3 all follow a general approach. The fibrinogen portion and the thrombin portion are printed as alternating layers. The number of layers printed is determined by the operator, but is typically in the range of 2-20. After the desired number of fibrinogen layers and thrombin layers are printed, the paste composition is printed on top of the layered fibrinogen / thrombin structure.

[0325] Taking the third part composition #7 (alginate and β-TCP) as an example, using a 3D printer, the fibrinogen component and the thrombin component (Table 1) were printed alternately from separate containers as follows. 3 The first layer of a fibrinogen component with a volume of (17.7 μL) was printed onto the print surface area. 3 A second layer of thrombin (17.7 μL) was printed on top of the previous layer of fibrinogen. A total of 8 layers of fibrinogen interlaced with 8 layers of thrombin were printed to create an alternating layered structure.

[0326] Then, 0.149cm 3 The third part composition #7 outlined in Table 3 of (149 μ L) is printed on the top of the alternating layered structure of fibrinogen and thrombin, and this material is printed again from a separate container. The resulting structure comprising three kinds of compositions printed on each other is referred to as the first macro layer (macrolayer). In this macro layer, the total ratio of fibrinogen composition to thrombin composition and composition #7 is about 1:1:1. In the macro layer, the fibrinogen layer and the thrombin layer react to form fibrin and strengthen the structure.

[0327] Additional macro layers are printed adjacent to and on top of the first macro layer in a sequential order according to the design file to construct Figure 1 Bone constructs outlined in .

[0328] The shear rate acting on the composition in the syringe tip during the printing process was determined to be 15 s -1 and 40s -1 .

[0329] Before the macroscopic layer of top is directly printed on the top of the macroscopic layer of bottom, the operator can carry out the other step of printing the substrate on the top of the macroscopic layer of bottom.The substrate is usually composed of a single layer of fibrinogen and a single layer of thrombin.The periphery of the substrate is upwardly protruding, to define the open space with the periphery that turns upward as the boundary.The substrate actually works as a nested body (nest) and looks similar to a nested body.The macroscopic layer of top is printed (as discussed above) in the open space limited in the substrate, and the periphery that turns upward provides a nesting function, which increases structural support to the macroscopic layer of top after it is printed out.

[0330] For the avoidance of any doubt, the concept of a substrate layer that prints a function similar to a nested body so that the other layers printed therein provide structure and support is a general concept that is applicable to all method steps of the present invention and can be combined with all method steps of the present invention. This concept should not be considered as being isolated from the specific embodiment discussed in the previous paragraph. The technician should understand the general applicability of the substrate layer or nested body layer to the method of the present invention.

[0331] Example 2

[0332] Compatibility of the composition of the present invention with human mesenchymal stem cells

[0333] The ability of the compositions of the present invention to provide a non-toxic environment to cells, proteins and other bioactive materials is highly advantageous. 3D printed bone constructs that promote cell survival and cell differentiation are highly desirable from a therapeutic perspective.

[0334] Human mesenchymal stem cells (hMSCs) are obtained from the bone marrow of human donors. hMSCs are extracted according to GMP specifications and then expanded in vitro in Dulbecco's modified Eagle's medium (DMEM) and 10% human serum B (hSERB). The cell culture medium is centrifuged and any excess liquid is removed. The resulting cells form a precipitate at the bottom of the centrifuge tube.

[0335] The hMSC precipitate was mixed with the composition #1, composition #2, composition #7 and composition #8 shown in Table 3. The hMSC precipitate was mixed with the relevant hydrogel, and the resulting mixture was added to the hydrated biocompatible inorganic material. The resulting paste was mixed manually (gently) to avoid any cell damage. The resulting compositions are summarized in Table 4, numbered #1', #2', #7' and #8' for ease of reference.

[0336]

[0337] *=75%hydroxyapatite(HA):25%β-TCP

[0338] Table 4

[0339] Composition #1', composition #2', composition #7' and composition #8' comprising hMSCs were mixed in vitro (without 3D printing) with the fibrinogen component and thrombin component of Table 1 at an approximate ratio of 1:1:1 by volume. The resulting mixture was cultured in the standard conditions discussed above (DMEM + 10% hSERB) for 7 days.

[0340] The CELLTITER-GLO 3D cell viability assay (Promega) was used to determine the number of viable cells in 3D cell cultures based on the quantification of ATP, a marker for the presence of metabolically active cells. After cell lysis, the luminescent signal obtained is proportional to the concentration of ATP present in the sample and, therefore, to the number of viable cells present in the culture.

[0341] Briefly, an equal volume of CELLTITER-GLO 3D reagent (Promega) was added to the cell culture volume and the contents were mixed vigorously for 5 minutes to induce cell lysis. The plate was incubated at room temperature for an additional 25 minutes to allow the luminescent signal to be recorded to stabilize.

[0342] Figure 2 The figure shows the results of the cell viability assay. Figure 2 It is evident that each of Composition #1', Composition #2', Composition #7' and Composition #8' showed an increase in ATP concentration on Day 3 and Day 7. Composition #7' depicted particularly promising results, although all compositions tested gave positive results. Therefore, it can be concluded that these compositions are non-toxic to hMSCs.

[0343] The compositions summarized in Table 4 were also subjected to a live / dead cell viability assay (Life Technologies), which is based on the simultaneous detection of live and dead cells by employing two probes that measure intracellular esterase activity (by Calcein AM probe) and plasma membrane integrity (by ethidium homodimer (EthD-1) probe), respectively. Nuclear contents were stained with Hoechst 33342 fluorescent dye. Fluorescence microscopy was used to visualize cellular features.

[0344] exist Figure 3In Figure 1, we see images from a cell viability assay captured with a 10X objective at day 0 and day 7. The image shows live hMSCs, dead hMSCs, and stained nuclei. At day 0, there is very little to note about any of the compositions. However, at day 7, the large shaded, speckled areas show a high concentration of cells that are alive when mixed with the composition of the invention. Thus, from Figure 3 It is clear from the present invention that the composition of the present invention is suitable for allowing cell adhesion and spatial cell distribution throughout the bone scaffold as well as promoting cell growth.

[0345] Example 3

[0346] Determination of the osteogenic potential of the composition of the invention

[0347] The osteogenic potency of the compositions summarized in Table 4 was determined by a cell differentiation assay protocol.

[0348] Composition #1', composition #2', composition #7' and composition #8' comprising hMSCs in Table 4 were mixed with the fibrinogen component and thrombin component of Table 1 in vitro (without 3D printing) at an approximate ratio of 1:1:1 by volume. The resulting mixture of this volume was supplemented with an equal volume of osteogenic differentiation medium (STEMPRO basal medium (90%) + STEMPRO Osteo supplement 10% from GIBCO). The osteogenic differentiation medium was replaced every 3 days.

[0349] On day 14, the alkaline phosphatase (ALP) activity of the composition / hMSC was tested using SIGMAFAST BCIP / NBT solution. The composition before differentiation culture did not show ALP staining (see day 0, Figure 4 ). However, on day 14, a large number of cells stained positive for ALP activity, as seen by Figure 4 This is shown in the figure of the silk structure stained on the 14th day. Figure 4 The figure shows a high number of ALP positive cells for each of Composition #1', Composition #2', Composition #7' and Composition #8' after 14 days of osteogenic culture. These findings positively demonstrate that the compositions of the present invention support successful differentiation / osteoinduction of hMSCs into osteoblasts. In addition, upon visual inspection on day 14, the cells showed osteocyte-like morphology.

[0350] All compositions tested promoted the differentiation of hMSCs into osteocytes.

[0351] Example 4

[0352] Printing of objects having defined geometries using the two-part composition of the present invention

[0353] Two-part compositions within the scope of the present invention are prepared according to Table 5. The first composition consists of the other of fibrinogen or thrombin as outlined above in Table 1, depending on whether the second composition comprises fibrinogen or thrombin. The second composition comprises the components outlined in Table 5.

[0354]

[0355] Table 5

[0356] like Figure 5 As shown in the diagram, the two-part compositions of the present invention do not 3D print with the level of control or accuracy of the three-part compositions of the present invention. However, promising results were obtained for Composition #A, Composition #B, and Composition #D, and Example 4 served as a proof of concept study that suitable bone constructs can be 3D printed with two-part compositions within the scope of the present invention and that accuracy can be improved with further effort and time.

Claims

1. A multi-part composition for three-dimensional printing of a bone composite material, the multi-part composition comprising: i) a first part comprising fibrinogen in a pharmaceutically acceptable carrier; ii) a second part comprising thrombin in a pharmaceutically acceptable carrier; as well as iii) a third part comprising a pharmaceutically acceptable hydrogel mixed with at least one biocompatible inorganic material, the at least one biocompatible inorganic material providing a source of at least one of calcium atoms and phosphorus atoms, wherein the third part has an apparent viscosity selected from the group consisting of: The viscosity of the sample was measured by a rotational viscometer at 25°C and a pressure of 1 atm in 0.1 s. -1 The shear rate ranges from 1Pa·s to 300Pa·s, and The viscosity of the sample was measured by a rotational viscometer at 25 °C and a pressure of 1 atm in 1 s. -1 The shear rate ranges from 1Pa·s to 100Pa·s, The third part is a separate composition such that the three parts of the multi-part composition are not mixed prior to printing by a three-dimensional printing device.

2. The composition of claim 1, wherein in the first part, the fibrinogen is at a concentration between 5 mg / mL and 200 mg / mL.

3. The composition according to any one of the preceding claims, wherein in the second portion, the thrombin is at a concentration between 25 IU / mL and 1500 IU / mL.

4. The composition of claim 1, wherein the third part of the multi-part composition has an apparent viscosity selected from the group consisting of: The viscosity of the sample was measured by a rotational viscometer at 25°C and a pressure of 1 atm in 0.1 s. -1 The shear rate ranges from 50 Pa·s to 250 Pa·s, and The viscosity of the sample was measured by a rotational viscometer at 25 °C and a pressure of 1 atm in 1 s. -1 The shear rate ranges from 2Pa·s to 50Pa·s.

5. The composition of claim 1, wherein the biocompatible inorganic material is selected from the group consisting of bioglass, tricalcium phosphate, single-phase hydroxyapatite, biphasic hydroxyapatite-tricalcium phosphate, natural bone powder, and combinations thereof.

6. The composition of claim 5, wherein the biocompatible inorganic material is selected from the group consisting of tricalcium phosphate, single-phase hydroxyapatite, biphasic hydroxyapatite-tricalcium phosphate, and combinations thereof.

7. The composition of claim 6, wherein the biocompatible inorganic material is β-tricalcium phosphate.

8. The composition of claim 7, wherein the beta-tricalcium phosphate has a relative humidity of 2.95 g / cm 3 and 3.15g / cm 3 The density between.

9. The composition of claim 7, wherein the beta-tricalcium phosphate has a relative humidity of 2.95 g / cm 3 and 3.10g / cm 3 The density between.

10. The composition according to any one of claims 7 to 9, wherein the β-tricalcium phosphate has a d90 particle size distribution of no greater than 180 μm.

11. The composition of claim 10, wherein the β-tricalcium phosphate has a d90 particle size distribution of no greater than 160 μm.

12. The composition of claim 7, wherein the β-tricalcium phosphate is characterized by an X-ray powder diffraction pattern comprising the following °2θ (d values) when obtained using a Cu tube anode with K-alpha radiation: ) at angles of 17.0 (5.2), 21.9 (4.1), 25.8 (3.45), 27.8 (3.2), 29.65 (3.0), 31.0 (2.9), 32.45 (2.75), 34.4 (2.6), 46.9 (1.9), 48.0 (1.9), 48.4 (1.9) and 53.0 (1.7).

13. The composition of claim 1, wherein the biocompatible inorganic material has an average particle size of less than 150 μm.

14. The composition of claim 13, wherein the biocompatible inorganic material has an average particle size of less than 100 μm.

15. The composition of claim 1, wherein the biocompatible inorganic material is hydrated with an albumin solution prior to mixing with the pharmaceutically acceptable hydrogel.

16. The composition of claim 1, wherein the pharmaceutically acceptable hydrogel is selected from the group consisting of hydrogels including hydrophilic polysaccharides, gelatin, and combinations thereof.

17. The composition of claim 1, wherein the pharmaceutically acceptable hydrogel is selected from the group consisting of alginate, hyaluronic acid, gelatin, and combinations thereof.

18. The composition of claim 17, wherein the pharmaceutically acceptable hydrogel comprises alginate.

19. The composition of claim 1, wherein the third portion has a ratio of biocompatible inorganic material:hydrogel of between 1 g-10 g of biocompatible inorganic material to 1 mL of hydrogel.

20. The composition of claim 1, wherein the multi-part composition further comprises a material having osteoconductive, osteoinductive and / or osteogenic properties.

21. The composition according to claim 20, wherein the material having osteoconductive, osteoinductive and / or osteogenic properties is selected from the group consisting of cells, proteins, growth factors and combinations thereof.

22. The composition of claim 21, wherein the multi-part composition further comprises human mesenchymal stem cells (hMSC).

23. The composition of claim 22, wherein the hMSCs are in a pharmaceutically acceptable hydrogel at 1*10 3 hMSCs to 5*10 10 The concentration of hMSCs was 24. A bone composite material obtainable from a multi-part composition according to any one of claims 1 to 23.

25. The bone composite material of claim 24, wherein the biocompatible inorganic material is at a concentration between 5% w / w and 60% w / w of the bone composite material.

26. A method for preparing a bone composite material, the method comprising the following steps: i) providing a three-dimensional printing device having a multi-part composition according to any one of claims 1 to 23; ii) printing the bone composite material according to the determined design; as well as iii) optionally incubating the bone composite material; The three-dimensional printing device prints to produce an alternating layered structure.

27. The method according to claim 26, wherein the three-dimensional printing device prints, i) a first layer comprising fibrinogen or thrombin; ii) a second layer comprising fibrinogen or thrombin; wherein if the first layer comprises fibrinogen, the second layer comprises thrombin, and vice versa, iii) repeating step i) and step ii) n times, wherein n≥1, to produce the alternating layered structure; iv) printing said third portion comprising said hydrogel and a biocompatible inorganic material on top of said alternating layered structure of step iii); and v) optionally repeating steps i) to iv) as needed to provide the bone composite material.

28. The method of claim 27, wherein the fibrinogen is printed first and the thrombin is printed second as a layer on top of the fibrinogen.

29. A method according to any one of claims 27 to 28, wherein: i) the first layer has a defined volume of X, ii) the second layer has a defined volume of Y, iii) step i) and step ii) are repeated n times, wherein n≥1, to produce said alternating layered structure having a volume nX+nY; and iv) said third part comprising said hydrogel and said biocompatible inorganic material is printed on top of said alternating layered structure of step iii), said third part having a volume Z.

30. The method of claim 29, wherein the volume X and volume Y are the same, such that the volume nX = nY.

31. The method of claim 29, wherein the ratio of Z to nX to nY (Z:nX:nY) is 0.60-1.0:1:

1.

32. The method of claim 29, wherein 2≤n≤10.

33. The method of claim 26, wherein the three-dimensional printing device prints; i) a first layer comprising the third part of the multi-part composition according to any one of claims 1 to 23; ii) a second layer comprising fibrinogen or thrombin; iii) a third layer comprising fibrinogen or thrombin; wherein if the second layer comprises fibrinogen, the third layer comprises thrombin, and vice versa, and iv) repeating step ii) and step iii) n times in sequence, wherein n≥1, to produce the alternating layered structure having fibrinogen and thrombin; and v) optionally repeating steps i) to iv) as needed to provide the bone composite material.

34. The method of claim 33, wherein: i) the first layer has a defined volume of A, ii) the second layer has a defined volume of B, iii) the third layer has a defined volume of C, iv) wherein step ii) and step iii) can be repeated n times, wherein n≥1, to produce a layered structure having a volume A+nB+nC.

35. The method of claim 34, wherein the ratio of A to nB to nC (A:nB:nC) is 1-5:1:

1.

36. The method of any one of claims 33 to 35, wherein 2≤n≤10.

37. A bone composite material obtainable by the method according to any one of claims 26 to 36.

38. Use of a multi-part composition according to any one of claims 1 to 23 for the manufacture of a synthetic bone composite material for treating bone injuries or bone defects.

39. A kit comprising a three-dimensional printer and a multi-part composition according to any one of claims 1 to 23.

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