WO2021232108A1 - Ceramic biofabrication - Google Patents

Abstract

Scaffold-based bone tissue engineering relies on the use of bioceramics, due to their inherent biocompatibility and bone bioactivity. However, fabrication of ceramic scaffolds may require harsh post-processing steps and toxic additives, which prevents construction in biological environments. Disclosed herein are biocompatible composite ceramic materials, which may be used as a rapidly solidifying ceramic ink, potentially for single-step extrusion of complex bone-like structures in the presence of live cells. The composites may utilise a bioceramic printing technique that facilitates omnidirectional generation of hierarchically structured bone analogues under mild conditions in the presence of live cells. A matrix suspension surrounding the printed architecture may be used to promote one or more of: robust cell adhesion, proliferation, and osteogenesis in proximity to the composite materials and possibly inks derived therefrom. The compositions may be used in high- resolution printing of ceramic structures within a cell-laden matrix. This may provide opportunities for "one-pot" generation of custom bone microenvironments for disease modelling and regenerative engineering, with scope for translation to in situ bone reconstruction during surgery.

Description

"CERAMIC BIOFABRICATION"

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority from Australian Provisional Patent Application No 2020901630 filed on 20 May 2020, the contents of which are incorporated herein by reference in their entirety.

TECHNICAL FIELD

The disclosure relates to scaffold-based bone tissue engineering relies, and the use of bio ceramics, due to their inherent biocompatibility and bone bioactivity. Also disclosed herein is the fabrication of ceramic scaffolds which may reduce or negate the need for harsh post-processing steps and/or toxic additives, which may prevent construction in biological environments.

BACKGROUND

Bone tissue has endogenous mechanisms for self-repair. Nevertheless, large fractures, known as segmental bone defects, can lead to poor treatment options and thus permanent damage. Bone defects can be caused either by trauma or disease, including infection, tumour resection, skeletal abnormalities, or conditions in which bone regeneration is compromised, such as necrosis and osteoporosis. About 100,000 non union fractures are diagnosed each year in the U.S. alone with major direct and indirect burden on healthcare systems. Bone defects above a critical size require painful extraction and delicate replacement of autografts or application of scaffolds designed to balance both the mechanical and biological requirements for the promotion of regrowth and for the avoidance of amputation. Despite the increased understanding of bone biology, adequate mimicry of the complex structure of bone with composite biomaterials remains elusive.

Attempts to mimic the bone microenvironment involve a myriad of traditional techniques including gas foaming, salt leaching, freeze-drying, and polymer template methods for the fabrication of scaffolds for bone regeneration. Recent advances in three dimensional printing have provided new opportunities for bone scaffold manufacturing, by either: embedding ceramic materials and cells in three dimensional soft hydrogels; or by seeding bone-progenitor cells on prefabricated ceramic scaffolds. Although soft hydrogels, such as collagen, hyaluronic acid, alginate and chitosan show good biocompatibility, they lack mechanical integrity and degrade quickly, especially when decorated with proteins. Moreover, when printed in air, hydrogel -based biomaterials rely on supported layer-by-layer additions which prevents accurate recreations of the irregular, delicate skeletal bone framework. While these hydrogels reflect aspects of the extracellular matrix in many tissues, they cannot recreate the highly calcified architecture of natural bone.

Bio ceramic-based scaffolds are known for use in bone regeneration due to similar chemical composition and mechanical properties, and to inherent bioactivity and osteoconductive properties. However, current fabrication approaches may be unable to accurately mimic the bone microenvironment due to the failure to replicate nanoscale complexity, and the artificial two dimensional stacked “log-pile” macro-architecture. Supersaturated mineral analogues have been developed to recapitulate the nanostructure, but these materials cannot be printed to provide bone-mimetic macro structures. Additive manufacturing techniques have been developed for printing bioceramic scaffolds including stereolithography, selective laser sintering, three dimensional printing, and direct-ink writing. These techniques can be generally classified as slurry-based or powder-based. In slurry-based techniques such as stereolithography and direct-ink writing, fine ceramic particles are dispersed in a liquid phase to form ink or paste and ultimately sintered to form the three dimensional construct, while in powdered-based techniques such as three dimensional printing and selective laser sintering, ceramic particles are spread over a printing bed and locally bonded by laser or a binder. In all of these approaches, cells must be seeded after scaffold fabrication. Although the invention will be described with reference to specific examples it will be appreciated by those skilled in the art that the invention may be embodied in many other forms. The skilled person will appreciate that the term "biocompatible" defines a two- way response, i.e. a body's response to the material and the material's response to the body. The biocompatibility of a medical device refers to the ability of the device to perform its intended function, with the desired degree of incorporation in the host, without eliciting any significant or long-term undesirable local or systemic effects in that host.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each claim of this application.

SUMMARY

Disclosed herein are bio ceramic materials, optionally in the form of granules, cements, pastes, putties, and slurries, along with associated biofabrication and bioprinting methods. The disclosure includes medical applications, but the person skilled in the art will be aware that alternative applications are available.

In a first aspect, disclosed herein is a composite biocompatible ceramic based material comprising particles of an inorganic salt of phosphoric acid, wherein the particles of phosphorous containing inorganic salts are dispersed in a mixture comprising an oil phase and a surfactant.

In one embodiment of the first aspect, the composite biocompatible ceramic- based material is designed for use as an ink, for example in an additive manufacturing process. In a second aspect, disclosed herein is an ink for additive manufacturing, the ink comprising a composite biocompatible ceramic-based material which when deposited in a yield-stress support matrix in the presence of biological materials allows free-form three dimensional printing of ceramic bone mimetic shapes integrated with the biological materials. In one embodiment of the second aspect, the composite biocompatible ceramic- based material is defined by the first aspect.

In a third aspect, disclosed herein is a method of making a composite biocompatible ceramic based material, the method comprising mixing an oil phase, a surfactant and an accelerator together to form a suspension, adding particles of an inorganic salt of phosphoric acid to the suspension, and homogenising the mixture to form a paste of the ceramic based material.

In one embodiment of the third aspect, the composite biocompatible ceramic- based material is defined by the first aspect.

In a fourth aspect, disclosed herein is a method of fabrication of a composite biocompatible ceramic material structure using an additive manufacturing process, the method comprising: depositing a composite biocompatible ceramic-based material in a yield-stress support matrix bath comprising a biological material, performing omnidirectional three-dimensional printing of a structure composed of the biocompatible ceramic material integrated with the biological material, wherein the yield-stress support matrix holds the printed ceramic material in place to obtain free-form printing of the structures.

In one embodiment of the fourth aspect, the composite biocompatible ceramic- based material is defined by the first aspect.

In a fifth aspect, disclosed herein is a bone like structure mimicking bone and formed by the method of the third or fourth aspect, using the composite biocompatible ceramic-based material of the first aspect, or the ink of the second aspect.

In a sixth aspect disclosed herein is a method of using the composite biocompatible ceramic-based material of the first aspect or the ink of the second aspect as a bone cement.

In a seventh aspect, disclosed herein is a method of encapsulating at least one biological molecule and/or bioactive molecule, the method comprising providing a composite biocompatible ceramic-based material of the first aspect or the ink of the second aspect, and at least one biological molecule and/or bioactive molecule, and allowing the composite biocompatible ceramic-based material or the ink to set.

In an eighth aspect disclosed herein is a method of promoting bone formation in a subject, the method comprising administering: a composite biocompatible ceramic- based material of the first aspect, optionally comprising at least one bioactive molecule and/or at least one biological molecule; or the ink of the second aspect, optionally comprising a bioactive molecule and/or a biological molecule, to the subject.

Disclosed herein is a technique which may utilise a chemically stabilised gelatin microsphere support bath, where the optimised yield-stress properties support the omnidirectional printing of a bone mineral-transforming ink in the presence of a biological material, for example live cells. This technique, labelled as ceramic omnidirectional bioprinting in cell-suspensions (COBICS), may provide solutions to the major challenges in generation of bone mimicked tissue engineering constructs mimicking bone microenvironment. COBICS may be capable of printing complex and biologically relevant architecture constructs without the need for sacrificial support materials, on-spot and without laborious post-processing steps which are two biggest challenges in additive manufacturing techniques of the bone mimetic construct. The ability to print nanostructured bone-mimetic ceramics within cell-laden biological materials in free-form with control over macro- and micro-architecture, can provide scope for complex bone mimicry and real-time bone reconstruction in clinical settings.

It will be appreciated that the embodiments of each aspect of the present disclosure may equally be applied to each other aspect, mutatis mutandis.

BRIEF DESCRIPTION OF DRAWINGS

Whilst it will be appreciated that a variety of embodiments of the invention may be utilised, in the following, we describe a number of examples of the invention with reference to the following drawings:

Figure 1 shows a possible mechanism for bone-ink nanoprecipitation and solidification.

Figure 2 shows a schematic demonstrating printing free-form ceramic inks in a suspension of microspheres with properties of a yield-stress fluid.

Figure 3 shows schematic of ceramic bioprinting in the presence of live cells.

Figure 4 shows: a top view of three dimensional printed construct after 7 days culture with mesenchymal stem cells (MSCs) (image B); viability staining of MSCs and osteoblast cells seeded on bone-ink and scanning electron microscopy of MSCs on bone- ink after 7 days (image C); and viability and cell shape analysis of MSCs cultured on ceramic ink, cell viability of human osteoblasts on ink, and MSCs viability embedded within the support bath and migration of MSCs toward bone-ink (image D).

Figure 5 shows image of direct bone-ink hand-printing in aqueous media and scanning electron micrographs demonstrating the nanostructured interface.

Figure 6 shows a comparison of storage modulus and apparent viscosity of ink as a function of time in humid and dry (inset) condition.

Figure 7 shows scanning electron microscopy (SEM) images showing α-TCP particles (top), the onset of hydrolysis and degradation of α-TCP particles in contact with the aqueous environment (middle) and formation of calcium-deficient hydroxyapatite crystals (bottom).

Figure 8 shows typical defects in ink filaments after extrusion at room temperature. Figure 9 shows a distribution of bovine serum albumin labelled with fluorescein isothiocyanate (FITC) in cross-section of scaffolds incorporated by submerging a sintered scaffold in the protein solution (top) or direct mixing of the protein with ink (bottom).

Figure 10 image A shows comparative drug release profiles of dexamethasone and ibuprofen loaded into bone-ink scaffolds and sintered hydroxyapatite scaffolds. Image B shows the compressive strength of printed bone-ink compared to cancellous bone and sintered scaffolds.

Figure 11 shows representative assembly of gelatin microspheres and ink filaments and modelling data.

Figure 12 shows size distribution and optical image of crosslinked gelatin microspheres by glutaraldehyde at fully hydrated state.

Figure 13 shows optimisation experiments for gelatin microsphere and cell concentration.

Figure 14 shows cell-microsphere size compatibility.

Figure 15 shows rheology results for a glutaraldehyde treated gelatin microsphere bath.

Figure 16 shows: fabrication of complex bone-mimetic architectures, photographs of a printed bone ink filament within a gelatin microsphere support bath and interfacial adhesion during nanocrystal formation (image A); a front view of the printing process in a 96-well cell culture plate (image B); and images C, D, E and F show examples of multiphase construct for potential application in osteochondral defect; generation of high-resolution bone mimicked constructs with hierarchical microstructure, such as human osseous labyrinth (D), trabecular bone (E) and helical haversian canal (F).

Figure 17 shows an effect of gelatin microsphere size on the deformation of ink filaments extruded through nozzles with different diameters.

Figures 18 shows: osteogenic gene expression (images A to D); and schematic representation of cells present in gelatin bath, far from the ink (E) and in proximity to the bone-ink (F). Representative images of cells stained for osteopontin (red in true images) in the vicinity of the bone-ink (F, H, J) and far from the bone-ink (E, G, I), either in expansion (G, H) or osteogenic medium (I, J), and Nuclei were counterstained with DAPI (blue in true images). Figure 19 shows examples of computer-generated three dimensional models (top) and corresponding three dimensional printed bone-like structures (bottom) through COB ICS technique

DETAILED DESCRIPTION General Definitions and Terms

With regards to the definitions provided herein, unless stated otherwise, or implicit from context, the defined terms and phrases include the provided meanings. In addition, unless explicitly stated otherwise, or apparent from context, the terms and phrases below do not exclude the meaning that the term or phrase has acquired by a person skilled in the relevant art. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims.

All publications discussed and/or referenced herein are incorporated herein in their entirety, unless described otherwise. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Throughout this disclosure, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e., one or more) of those steps, compositions of matter, groups of steps or groups of compositions of matter. Thus, as used herein, the singular forms “a”, “an” and “the” include plural aspects unless the context clearly dictates otherwise. For example, reference to “a” includes a single as well as two or more; reference to “an” includes a single as well as two or more; reference to “the” includes a single as well as two or more and so forth. Those skilled in the art will appreciate that the disclosure herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the examples, steps, features, methods, compositions, formulations, and processes, referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

The term “and/or”, e.g., “X and/or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning. Unless otherwise indicated, the terms “first,” “second,” etc. are used herein merely as labels, and are not intended to impose ordinal, positional, or hierarchical requirements on the items to which these terms refer. Moreover, reference to a “second” item does not require or preclude the existence of lower-numbered item (e.g., a “first” item) and/or a higher-numbered item (e.g., a “third” item).

As used herein, the phrase “at least one of’ or “one or more of’ when used with a list of items, means different combinations of one or more of the listed items may be used and only one of the items in the list may be needed. The item may be a particular object, thing, or category. In other words, “at least one of’ means any combination of items or number of items may be used from the list, but not all of the items in the list may be required. For example, “at least one of item A, item B, and item C” may mean item A; item A and item B; item B; item A, item B, and item C; or item B and item C. In some cases, “at least one of item A, item B, and item C” may mean, for example and without limitation, two of item A, one of item B, and ten of item C; four of item B and seven of item C; or some other suitable combination.

It is to be appreciated that certain features that are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any sub-combination.

Throughout the present specification, various aspects and components of the invention can be presented in a range format. The range format is included for convenience and should not be interpreted as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range, unless specifically indicated. For example, description of a range such as from 1 to 5 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 5, from 3 to 5 etc., as well as individual and partial numbers within the recited range, for example, 1, 2, 3, 4, 5, 5.5 and 6, unless where integers are required or implicit from context. This applies regardless of the breadth of the disclosed range. Where specific values are required, these will be indicated in the specification.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. Throughout this specification, the term "consisting essentially of' is intended to exclude elements which would materially affect the properties of the claimed composition, method or process.

The terms "comprising", "comprise" and "comprises" herein are intended to be optionally substitutable with the terms "consisting essentially of, "consist essentially of', "consists essentially of^", "consisting of^", "consist of' and "consists of, respectively, in every instance.

Herein the term “about” encompasses a 10% tolerance in any value or values connected to the term.

Herein “weight %” may be abbreviated to as “wt%” or “wt.%”

Composite biocompatible ceramic based material

Disclosed herein is an alternative approach to traditional ceramic-based scaffold fabrication. In one embodiment a bio-ceramic material formulation induces rapid crystallisation in an aqueous environment, with the ability to integrate bioactive molecules and/or to generate the bone mimic structures.

Also disclosed herein is a composite biocompatible ceramic based material comprising particles of an inorganic salt of phosphoric acid, wherein the particles of the inorganic salt of phosphoric acid are dispersed in a mixture comprising an oil phase and a surfactant.

The particles may be in a range of about 0.5 to about 5 microns, for example a range of about: 1 to 5 microns, 2 to 5 microns, 3 to 5 microns, or 4 to 5 microns. In another embodiment the particle are at least about, 0.5 microns, 1 micron, 2 microns, 3 microns, 4 microns or 5 microns, or a mixture thereof. In yet another embodiment the particle are less than about, 0.5 microns, 1 micron, 2 microns, 3 microns, 4 microns or 5 microns, or a mixture thereof.

In one embodiment the particles of the inorganic salt of phosphoric acid are homogeneously dispersed in the mixture comprising an oil phase and a surfactant. In one embodiment the biocompatible ceramic based material is designed such that it may be used as an ink in an additive manufacturing process. The composite biocompatible ceramic based material may be formulated as an ink, for example a ceramic-ink, bone ink or bio-ink. Herein, with regards to the aspects and embodiments, the terms “ink”, “ceramic ink”, “bone ink” and “bio-ink” may be used interchangeably, unless dictated specifically or by context. Herein “ink” may be regarded as a flowable material in the form of or comprising a liquid, or a liquid suspension comprising granules, cements, pastes, putties and slurries.

Herein “ceramic-ink” may be regarded as an ink containing inorganic ions that form compound crystals upon setting.

Herein “bone-ink” may be regarded as an ink where the set material approximates the physical structure and composition of natural bone.

Herein “bio-ink” may be regarded as an ink comprising biological materials and/or an ink that is biological in structure and composition upon setting.

Inorganic salt of phosphoric acid

The inorganic salt of phosphoric acid may be in an appropriate form known in the art. In one embodiment the inorganic salt of phosphoric acid is in the form of a powder, agglomerate or granule.

As used herein, the term "salt" refers to acid or base salts of the compounds or materials and their use in the applications and methods defined herein. In one embodiment the salts are pharmaceutically acceptable salts, and are non-toxic. Additional information on suitable pharmaceutically acceptable salts can be found in Remington: The Science and Practice of Pharmacy, 21^st Edition; Lippincott Williams & Wilkins: Philadelphia, PA, 2005, which is incorporated herein by reference.

Pharmaceutically acceptable salts of the acidic compounds of the present invention are salts formed with bases, namely cationic salts such as alkali and alkaline earth metal salts, such as sodium, lithium, potassium, calcium, magnesium. In one embodiment the salt is a calcium salt.

Examples of salts include, but are not limited to: calcium phosphosilicate, calcium phosphate, hydroxyapatite, alpha-tricalcium phosphate (α-TCP), dicalcium phosphate, alpha-tricalcium phosphate, beta-tri calcium phosphate (β-TCP), tetracalcium phosphate, amorphous calcium phosphate, octacalcium phosphate, BIOGLASS, fluoroapatite, chlorapatite, oxyapatite, magnesium-substituted tricalcium phosphate, carbonate hydroxyapatite, substituted forms of hydroxyapatite (e.g, hydroxyapatite derived from bone may be substituted with other ions such as fluoride, chloride, magnesium sodium, potassium, etc.), metal atoms doped calcium phosphates or calcium silicates, interstitially or substitutionally, or combinations or derivatives thereof. In one embodiment the inorganic salt of phosphoric acid is α-TCP or β-TCP.

In one embodiment the inorganic salt of phosphoric acid in the composite biocompatible ceramic based material is α-TCP, which may be in the form of a powder.

In one embodiment the α-TCP powder may be synthesised by mixing calcium carbonate and calcium hydrogen phosphate in a relative proportion to influence the compound crystal stoichiometry. For example the calcium carbonate and calcium hydrogen phosphate may be in a molar ratio range selected from but not limited to about: 0.5:2, 1:2; 1:1; or 1.5:2. In one embodiment the range is about 1:2 on a molar ratio basis.

Oil phase

The oil phase may comprise a single oil or a mixture of oils. The chosen oil phase may be appropriately selected by a person skilled in the art.

In one embodiment, no specific limitation placed on the oil phase. In one embodiment, any type of oil-based organic biocompatible material can be potentially used. In one embodiment the oil phase comprises a non-aqueous liquid, for example an organic solvent. In one embodiment the oil phase may comprise one or more compounds selected from, but not limited to: glycerol, an unsaturated fatty acid, a triglyceride, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain(C₈-C₁₂)mono, di, and/or tri-glycerides, polyoxy ethylated glyceryl fatty acid esters, fatty alcohols, polyglycolised glycerides, saturated polyglycolised C₈-C₁₀ glycerides, vegetable oils, silicone oil, and mixtures thereof.

In one embodiment the oil phase comprises glycerol.

Surfactant

At least one surfactant may be used for the composite biocompatible ceramic based material.

The surfactant may be an appropriate surfactant known in the art. Examples of surfactants include, but are not limited to: anionic, cationic, zwitterionic, non-ionic, hydrophobic, hydrophilic, and mixtures thereof. Examples of possible surfactants include, but are not limited to: fatty amine salts; fatty alkyl quaternary amines including primary, secondary, and tertiary amines; ester amines and the corresponding ethoxylated ester amines; sodium and potassium salts of straight-chain fatty acids, polyoxyethylenated fatty alcohol carboxylates, linear alkyl benzene sulfonates, alpha olefin sulfonates, sulfonated fatty acid methyl ester, arylalkanesulfonates, sulfosuccinate esters, alkyldiphenylether(di)sulfonates, alkylnaphthalenesulfonates, isoethionates, alkylether sulfates, sulfonated oils, fatty acid monoethanolamide sulfates, polyoxyethylene fatty acid monoethanolamide sulfates, aliphatic phosphate esters, nonylphenolphosphate esters, sarcosinates, fluorinated anionics, anionic surfactants derived from oleochemicals, sorbitan monostearate, polyoxyethylene ester of rosin, polyoxyethylene dodecyl mono ether, polyoxyethylene-polyoxypropylene block copolymer, polyoxyethylene monolaurate, polyoxyethylene monohexadecyl ether, polyoxyethylene monooleate, polyoxyethylene mono(cis-9-octadecenyl)ether, polyoxyethylene monostearate, polyoxyethylene monooctadecyl ether, polyoxyethylene dioleate, polyoxyethylene distearate, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan trioleate, polyoxyethylene sorbitan tristearate, polyglycerol ester of oleic acid, polyoxyethylene sorbitol hexastearate, polyoxyethylene monotetradecyl ether, polyoxyethylene sorbitol hexaoleate, fatty acids, tail-oil, sorbitol hexaesters, ethoxylated castor oil, ethoxylated soybean oil, rapeseed oil ethoxylate, ethoxylated fatty acids, ethoxylated fatty alcohols, ethoxylated polyoxyethylene sorbitol tetraoleate, glycerol and polyethylene glycol mixed esters, alcohols, polyglycerol esters, monoglycerides, sucrose esters, alkyl polyglycosides, polysorbates, fatty alkanolamides, polyglycol ethers, derivatives of any thereof, and combinations of any thereof.

In one embodiment the surfactant is a non-ionic surfactant.

In another embodiment the non-ionic surfactant comprises polyoxyethylenesorbitan monooleate.

Accelerator

In some forms the composite biocompatible ceramic based material further comprises an accelerator. In one embodiment an accelerator regulates the pH (for example increasing the pH to a more basic environment), and/or releases Ca, P, and Na or K ions to the microenvironment of a reaction that facilitate formation of apatite crystals. In one embodiment any salt that can provide a favourable condition for apatite formation, for example increasing pH and/or increasing ion concentration could be potentially used as the accelerator. Examples of compounds which may be used as an accelerator include, but are not limited at least compounds which is a salt comprising Ca, P, Na, K ions that dissolves in aqueous solutions and increases the pH and releases the ions causing ion supersaturation at the microenvironment of reaction.

In one embodiment at least one accelerator is ammonium phosphate dibasic (APD). In another embodiment at least one accelerator is sodium phosphate dibasic.

In one embodiment, the composite biocompatible ceramic based material may have: a) at least about 30 wt.%; or at least about 35 wt.%; or at least about 40 wt.%; or at least about 45 wt.%; or at least about 50 wt.%; or at least about 55 wt.%; or at least about 60 wt.%; or at least about 65 wt.%; or at least about 70 wt.%; or at least about 75 wt.%; or at least about 80 wt.% of the inorganic salt of phosphoric acid; or between about 30 and 70 wt.%; between about 35 and 70 wt.%; between about 40 and 70 wt.%; between about 45 and 70 wt.%; between about 50 and 70 wt.%; between about 55 and 70 wt.%; between about 60 and

70 wt.%; between about 65 and 70 wt.%; between about 30 and 65 wt.%; between about 30 and 60 wt.%; between about 30 and 55 wt.%; between about 30 and 50 wt.%; between about 30 and 45 wt.% of the inorganic salt of phosphoric acid, in a mixture comprising: b) at least about 20 wt.%; or at least about 25 wt.%; or at least about 30 wt.%; or at least about 35 wt.% of the oil phase; between about 20 and 35 wt.%; between about 20 and 30 wt.%; between about 20 and 25 wt.% between about 25 and 35 wt.%; or between about 30 and 35 wt.% of the oil phase; c) at least about 5 wt.%; or at least about 10 wt.% of the surfactant; or about 5 to 10 wt.% of the surfactant; and optionally d) at least about 3 wt.%; or at least about 4 wt.%; or at least about 5 wt.%; or at least about 6 wt.%; or at least about 7 wt.%; or at least about 8 wt.% of an accelerator; or between about 3 and 8 wt.%; between about 4 and 8 wt.%; between about 5 and 8 wt.%; between about 6 and 8 wt.%; between about 7 and 8 wt.%; between about 3 and 6 wt.%; between about 3 and 5 wt.%; between about 3 and 4 wt.% of an accelerator.

In one embodiment, the composite biocompatible ceramic based material may have between about 50 and 70 wt.% of particles of the inorganic salt of phosphoric acid dispersed (optionally homogenously) in a mixture of about 20 to 35 wt.% of the oil phase, about 5 to 10 wt.% of the surfactant and about 3 to 8 wt.% of an accelerator. Inks

Disclosed herein is an ink suitable for additive manufacturing, the ink composed of a composite biocompatible ceramic-based material, as described herein, which when deposited in or on a yield-stress support matrix in the presence of at least one biological material and/or bioactive molecule, and allows free-form three dimensional printing of ceramic bone mimetic shapes integrated with the biological materials.

In one embodiment, the composite biocompatible ceramic-based material used in the formation of the ink comprises particles of an inorganic salt of phosphoric acid; the particles of the inorganic salt of phosphoric acid being dispersed in a mixture (optionally homogeneously), comprising an oil phase and a surfactant.

In one embodiment, the ink is composed of a material which when deposited in or on a yield stress support matrix forms free-form three-dimensional printing of simple and complex ceramic bone mimetic shapes optionally integrated with at least one bioactive molecule and/or biological molecule. In an embodiment the ink, may be referred to as a: “ceramic ink”, “bone-ink” or

“bio-ink”.

In one embodiment the ink is a calcium phosphate-based formulation that quickly solidifies in an aqueous medium. In Figure 1, 10 depicts the mechanism for the bone- ink nanoprecipitation and solidification wherein: step (i) is hydrolytic surface degradation of 12 α-TCP as glycerol 11 is replaced with water 13; step (ii) is Ca-P nucleation and growth catalysed by ammonium phosphate dibasic (APD); and step (iii) is polyoxyethylenesorbitan monooleate (PS) directs nanocrystal growth and crystal entanglement leading to formation of hydroxyapatite (HA) crystal 14 formation due to mechanical interlocking.

The ink may takes advantage of the setting mechanism of pre-mixed calcium phosphate cements in aqueous solutions. Pre-mixed calcium phosphate cements may consist of a mixture of α-TCP powder as the main component and a non-aqueous carrier to prevent its setting during storage. However, the main disadvantages of pre-mixed cements are that the onset and setting time is drastically delayed, and the carrier phase/s or other additives are toxic to cells. Yield stress support matrix

In one more embodiment, a composite biocompatible ceramic-based material which when deposited in a yield-stress support matrix mixed in the presence of biological materials forms free-form three-dimensional printing of simple and complex ceramic bone mimetic shapes integrated with the optional bioactive molecules. The yield stress support matrix is a material that behave like a solid material and becomes fluid when shear forces are applied to the material.

In some embodiments the yield-stress support matrix may be a suspension, such as suspension of microspheres. The yield support matrix may be a natural or synthetic a hydrogel, a hydrogel forming polymer (for example a homopolymer or a copolymer), a gelator, or a mixture thereof. The yield-stress support matrix, optionally in the form of microspheres, may comprise at least one material selected from: gelatin, gelatin methacryloyl (GelMA), one or more polymers selected from: polysaccharides, nucleic acids, carbohydrates, proteins, polypeptides, poly(a-hydroxy acids), poly(lactones), poly(amino acids), poly(anhydrides), poly(orthoesters), poly(anhydride-co-imides), poly(orthocarbonates), poly(a-hydroxy alkanoates), poly(dioxanones), poly(phosphoesters), poly(L-lactide) (PLLA), poly(D,L-lactide) (PDLLA), polyglycolide (PGA), poly(lactide-co-glycolide (PLGA), poly(L-lactide-co-D, L- lactide), poly(D,L-lactide-co-trimethylene carbonate), polyhydroxybutyrate (PHB), poly(s-caprolactone), poly(δ-valerolactone), poly(Υ-butyrolactone), poly(caprolactone), polyacrylic acid, polycarboxylic acid, poly(allylamine hydrochloride), poly(diallyldimethylammonium chloride), poly(ethyleneimine), polypropylene fumarate, polyvinyl alcohol, polyvinylpyrrolidone, polyethylene, polymethylmethacrylate, carbon fibres, poly(ethylene glycol), polyethylene oxide), poly(vinyl alcohol), poly(vinylpyrrolidone), poly(ethyloxazoline), poly(ethylene oxide)- co-poly(propylene oxide) block copolymers, poly(ethylene terephthalate)polyamide, and copolymers thereof; a homo- or co-polymer having one more monomers selected from the group consisting of acrolein potassium, (meth)acrylamides, (meth)acrylic acid and salts thereof, (meth)acrylates, acrylonitrile, ethylene, ethylene glycol, ethyleneimine, ethyleneoxide, styrene sulfonate, vinyl acetate, vinyl alcohol, vinyl chloride, and vinylpyrrolidone); a polyphenolic complexing agent selected from gallotannins, ellagitannins, taragallotannins, caffetannins, proanthocyanidins, catechin, epicatechin, chlorogenic acid, and arbutin; an agent selected from alginic acid, arabic gum, guar gum, xantham gum, gellan gum, gelatin, chitin, chitosan, carobpol, chitosan acetate, chitosan lactate, chondroitin sulfate, N,O-carboxymethyl chitosan, a dextran, fibrin glue, glycerol, hyaluronic acid, sodium hyaluronate, a cellulose, a glucosamine, a proteoglycan, a starch, lactic acid, a pluronic, sodium glycerophosphate, collagen, glycogen, a keratin, silk; and mixtures thereof.

In one embodiment the yield-stress support matrix comprises gelatin microspheres. In another embodiment the yield-stress support matrix may be a gel such as a hydrogel.

In yet another embodiment the yield-stress support matrix comprises gelatin methacryloyl (GelMA).

In some embodiments the yield-stress support matrix is a suspension of gelatin microspheres wherein the gelatin microspheres may dissolve at about 37 °C, and optionally printed structures may be harvested directly. The gelatin microspheres may be hydrated in cell culture media, and may allow the formation of a homogeneous bath. The yield-stress support matrix may be optimised and chemically stabilised.

The materials disclosed herein and/or the support yield matrix may be stabilised may be obtained by an appropriate method known in the art. The inclusion and/or modification of any polymerisable groups; enzyme-mediated stabilisation; and/or orthogonal molecular coupling reagents integrated within and without the support matrix may be used to stabilise the materials disclosed herein, including optionally the support yield matrix. To stabilise the yield-stress support matrix, a chemical crosslinker may be added.

The chemical crosslinker may be glutaraldehyde, genipin, lithium phenyl-2,4, 6- trimethylbenzoylphosphinate (LAP), 2-hydroxy-4'-(2-hydroxyethoxy)-2- methylpropiophenone (such as Irgacure 2959 from Sigma) or 2',4',5',7'- tetrabromofluorescein (such as Eosin-Y from Sigma), or the addition of a salt solution. The compound may be added to freshly made microspheres.

In one embodiment LAP is used. In one example the LAP is exposed to 395 nm light at 2 mW/cm², and could be used for photocrosslinking GelMa support matrices.

Methods of synthesis Disclosed herein is a method of making a composite biocompatible ceramic based material and/or ink as described herein. The method may comprise mixing an oil phase, a surfactant and optionally an accelerator together to form a suspension, adding particles of an inorganic salt of phosphoric acid to the suspension, and preparing a mixture (optionally homogenising the mixture) to form a composition, optionally in the form of a paste, of the ceramic based material.

Herein the biocompatible ceramic based material or ink may be in and/or manufactured in the form of a liquid suspension. Examples of liquid suspensions includes: granules, cements, pastes, putties, and slurries.

Also disclosed herein is a method of fabrication of a composite biocompatible ceramic material structure using an additive manufacturing process, the method comprising: depositing a composite biocompatible ceramic-based material or an ink, as defined herein, in a yield-stress support matrix bath comprising a biological material and/or bioactive molecule, performing omnidirectional three-dimensional printing of a structure composed of the biocompatible ceramic material integrated with the biological material and/or bioactive molecule, wherein the yield-stress support matrix holds the printed ceramic material in place to obtain free-form printing of the structures.

In some embodiments, the fabrication may be performed at room temperature, for example at about 20, 21, 22, 23, 24 or 25 °C.

In some embodiments the fabrication may be performed at a temperature of about: 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40°C.

In some embodiments a temperature close to physiological conditions may be used. The fabrication may be performed at a temperature of about 35, 36, 37 or 38°C

Additives/Excipients

The materials herein may be tailored for specific applications, thereby dictating the need for additional excipients. In one embodiment the materials comprise one or more bioactive materials and/or biological materials as defined herein.

In some embodiments the bioactive material is a pharmaceutical drug. In one embodiment the bioactive material is an anti-inflammatory medication, a cytokine, a growth factors, large and small bioinstructive/potent biomolecules, proteins, a peptide, or a mixture thereof, added to the biocompatible ceramic material, optionally before the step of printing. The pharmaceutically active compound may be: bactericidal/anti microbial, angiogenic, osteogenic, chondrogenic, and/or tenogenic. Examples of anti- inflammatory medication includes, but is not limited to: bisphosphonates, corticosteroids such as dexamethasone and non-steroidal anti-inflammatory drugs such as ibuprofen.

In some embodiments a bioactive molecule and/or biological material is added to the biocompatible ceramic material before the step of printing. In one embodiment, one or more bioactive molecules may comprise soluble or insoluble structural proteins, growth factors, cytokines, fluorescein isothiocyanate labelled bovine serum albumin (FITC-BSA), and mixtures thereof.

Biological Materials and Bioactive Materials

The inks, composite biocompatible ceramic-based material and/or products thereof may comprise at least one bioactive material and/or biological material.

Herein the terms “biological material” and “biological molecule” may be used interchangeably unless dictated specifically or by context. The terms includes matter that has come from a once-living organism, or a chemical substance present or produced in a living organism.

Herein the terms “bioactive material” and “bioactive molecule” may be used interchangeably unless dictated specifically or by context. The terms refer to molecules or materials having a biological effect on a eukaryotic organism or cell (e.g., a mammal such as human) when introduced into the human organism or cell. Bioactive molecules or materials may include, but are not limited to, therapeutic nucleic acids, therapeutic polypeptides, and therapeutic small molecule drugs.

Herein, the biological material or bioactive material may be selected from, but not limited to: live cells, proteins, spheroids, organoids, proteoglycans, nucleic acids, oligosaccharides, biological small molecules, imaging reagents, biomolecules, drugs, enzymes, antibodies, targeting ligands, ligand, polynucleotides, siRNA, RNA, RNA constructs, DNA, DNA constructs, vectors, plasmids, amino acids, carbohydrates, catalysts, whole cells, cell fragments, micelles, liposomes, hydrogels, peptides, soluble or insoluble proteins, growth factors, cytokines..

One or more biological material may be an antibody. Herein “antibody” or “antibodies” shall be taken to encompass a protein that comprises a variable region made up of one or more immunoglobulin chains. As used herein, the term “antibody” is also intended to include formats other than full-length, intact or whole antibody molecules, such as Fab, F(ab')2, and Fv which are capable of binding to an epitopic determinant. These formats may be referred to as antibody “fragments”. These antibody formats retain some ability to selectively bind to a target protein One or more biological material may be a polynucleotide. The term “polynucleotide” as used herein shall be taken to encompass DNA, RNA, an antisense polynucleotide, a ribozyme, an interfering RNA, a siRNA, a microRNA, and any other polynucleotide known in the art. The polynucleotide may encode a protein or a functional RNA (such as an interfering RNA). Thus, the polynucleotide may be a polynucleotide vector or plasmid.

Examples of bioactive materials may include: diagnostic drugs and reagents; electrolytes; skeletal muscle relaxants; analgesic, antipyretic, and/or anti-inflammatory drugs; anti-infectives; enzymes; nutrients and associated substances, or a mixture thereof. Herein the term "targeting ligand" refers to a molecule that binds to or interacts with a target molecule. Typically the nature of the interaction or binding is non-covalent, e.g., by hydrogen, electrostatic, or van der Waals interactions, however, binding may also be covalent.

The term “ligand”, as used herein, refers to compounds which target biological markers. Examples of ligands include, but are not limited to: proteins, peptides, antibodies, antibody fragments, saccharides, carbohydrates, glycans, cytokines, chemokines, nucleotides, lectins, lipids, receptors, steroids, neurotransmitters, cluster designation/differentiation markers, imprinted polymers, and the like.

Examples of imaging reagents include, but are not limited to: luciferase; fluorescently labelled dyes and antibodies, contrast agents (including: iopamidol, iohexol and ioxilan); barium sulfate; MRI contrast agents (for example: gadolinium, iron oxide and manganese based imaging agents); Indocyanine green (ICG), and mixture thereof.

Herein, the term “peptide” is intended to mean any polymer comprising amino acids linked by peptide bonds. The term “peptide” is intended to include polymers that are assembled using a ribosome as well as polymers that are assembled by enzymes (i.e., non-ribosomal peptides) and polymers that are assembled synthetically. In various embodiments, the term “peptide” may be considered synonymous with “protein,” or “polypeptide”.

Examples of amino acids include, but are not limited to: alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.

Examples of peptides include, but are not limited to: polypeptides comprising: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids selected from: alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.

Here the term “protein” refers to a sequence of amino acids for which the chain length is sufficient to produce the higher levels of tertiary and/or quaternary structure. The protein may have a molecular weight in a range of about 300 Da to about 150 kDa. The protein may have a molecular weight of greater than 150 kDa, or a molecular weight less than 300 Da.

In one embodiment the biological material comprises live cells. Examples of cells includes, but is not limited to: bone cells, osteoblasts, osteoprogenitor cells, periostal cells, endothelial cells, fibroblasts, connective tissue cells, muscle cells, fat cells, cartilage cells, tendon cells, vascular cells, blood cells, progenitor cells, mesenchymal stem cells (including stromal/stem cells derived from bone marrow adipose tissue, cartilage, skin, dental tissue, endometrium, menstrual blood, umbilical vein tissue, amniotic material, placenta, Wharton’s jelly and cord blood), epithelial cells, somatic cells, reprogrammed cells (including induced pluripotent stem cells), diseased cells, and mixtures thereof. Cells used in the materials disclosed herein (or the formation of said materials), may be immortalised commercial cell lines, or patient derived primary cells.

In one embodiment the composite biocompatible ceramic-based material as describe herein is designed to be used as ink and can incorporate a model protein, such fluorescein isothiocyanate labelled bovine serum albumin (FITC-BSA). The FITC-BSA may be incorporated into the ink prior to printing.

In another embodiment the composite biocompatible ceramic-based material may be designed to be used as ink and comprise at least one bioactive material which is a pharmaceutical drug. In one example the pharmaceutical drug may be an anti- inflammatory drug. One or more pharmaceutical drugs, for example one or more anti- inflammatory drugs, may be incorporated into the ink prior to printing. Uses

Disclosed herein is a composite biocompatible ceramic-based material which may be used as an ink in an additive manufacturing process, for example three-dimensional printing, wherein the material comprises, particles of an inorganic salt of phosphoric acid, the particles of inorganic salt of phosphoric acid being dispersed (optionally homogenously), in a mixture of an oil phase and a surfactant. In one embodiment, the materials disclosed herein may be used without printing.

Also disclosed herein is a method of using the composite biocompatible ceramic- based material or ink, as defined herein as a bone cement. The bone cement may be used to bond two or pieces of bone together and/or bond one or more synthetic materials to bone. The materials may be used as part of a procedure involving an artificial joint.

Also disclosed herein is a method of encapsulating a biological material and/or bioactive molecule as defined herein, the method comprising providing a composite biocompatible ceramic-based material or an ink as defined herein, and a biological material and/or a bioactive molecule, and allowing the composite biocompatible ceramic-based material or the ink to set.

The compositions (for example inks or biocompatible ceramic-based material), disclosed herein may be administered to a “patient” or “subject”, for example as a bone cement, or following the formation of a bone like structure. As used herein, the terms “patient” and "subject" refer to animals such as mammals, including, but not limited to, primates (e.g. humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice and the like. In certain embodiments, the subject is a human.

Herein, a bone like structure mimicking bone may be formed by a method disclosed herein, using a composite biocompatible ceramic-based material as defined herein, or an ink as defined here.

The bone like structure may be a high resolution construct with a hierarchical microstructure. For example, a multiphasic construct with hierarchical microstructure. The construct with hierarchical microstructure may be an irregular bone structure mimicking, for example one of: a human osseous labyrinth, a trabecular bone or a helical haver si an canal.

The bone like structure may be integrated with at least one bioactive molecule and/or biological molecule as defined herein.

The bone like structure may be integrated with biological cells and/or one or more bioactive molecule. For example, the bioactive molecule may be a protein, such as fluorescein isothiocyanate labelled bovine serum albumin (FITC-BSA).

In one embodiment, the bone like structure further comprises one or more drugs supporting sustained drug release. For example the drugs may be anti-inflammatory, such as dexamethasone and ibuprofen. Herein the three dimensional printing may be free-form, in air, in aqueous solution, in granular media, and using techniques like jet printing and extrusion, or an appropriate instrument may be used. In one embodiment robocasting may be used. In another embodiment bio-printers, including extrusion based bio-printers may be used.

In an embodiment the ceramic based material the setting time is dependent on the actual composition of the material. The setting time may be less than 24 hours. In some instances delaying solidification may be advantageous, for example when cells and/or another biological material are included in the material.

In another embodiment the ceramic based material is a rapidly solidifying ceramic ink for single-step extrusion of complex bone-like structures in the presence of a biological material such as live cells. In one embodiment “rapidly solidifying” relates to a timeframe of about 1 minute to about 6 minutes.

In one embodiment, the ceramic based material initially solidifies in: about 1 minute or less; about 2 minutes or less; about 3 minutes or less; about 4 minutes or less; about 5 minutes or less; about 6 minutes or less; about 8 minutes or less; about 9 minutes or less; or about 10 minutes or less. In another embodiment, the ceramic based material is set in: about 10 minutes or less; about 11 minutes or less; about 12 minutes or less; about 13 minutes or less; about 14 minutes or less; about 15 minutes or less; about 16 minutes or less; about 17 minutes or less; about 18 minutes or less; about 19 minutes or less; about 20 minutes or less.

The ink, biocompatible ceramic-based material or a bone like structure formed from a composition disclosed herein may be used to promote bone formation. For example to promote bone formation at a specific site of injury and/or due to a localised condition.

Herein the term "site of injury and/or localised condition" refers to a specific location in a subject's body that is in need of treatment by a method utilising the materials and compositions defined herein. For example, the injury can be a fracture and the localised condition can be a disease state (such as osteoporosis, etc.) that is limited to a particular location in the subject's body, such as a particular bone, joint, digit, hand, foot, limb, spine, head, torso, etc. In some embodiments, the site of injury or localised condition is a surgical implantation site.

Herein, the terms "treat," "treating," and "treatment" refers to any indicia of success in the treatment or amelioration of an injury, pathology, condition, or symptom pain), including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the symptom, injury, pathology or condition more tolerable to the patient; decreasing the frequency or duration of the symptom or condition, or, in some situations, preventing the onset of the symptom or condition. The treatment or amelioration of symptoms can be based on any objective or subjective parameter; including, e.g., the result of a physical examination.

Herein, the term "bone fracture" refers to bone that has been cracked, fractured, or broken in one or several locations along the bone. In some embodiments, the term "bone fracture" also includes a segment of the bone missing.

Herein, the term "promoting bone formation" refers to stimulating new bone formation, growing bone across a joint or gap, enhancing or hastening bone formation, and/or increasing bone density or bone mineral content. In some embodiments, the compositions and materials disclosed herein (such as biocompatible ceramic based material and/or ink), promotes bone formation if it increases the amount of bone in a sample by at least about” 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, or more relative to a control sample (e.g., a sample that has not been contacted with a materials as defined herein).

The materials and compositions disclosed here in may further comprise any one of: adjuvant, carrier, excipient, glidant, diluent, preservative, surfactant, dispersing agent, stabiliser, or a mixtures thereof, being acceptable for use in humans or an animal.

Disclosed herein is a method of making a composite biocompatible ceramic-based material which may be used as an ink in an additive manufacturing process wherein the method of making a composite biocompatible ceramic based material comprises mixing an oil phase, a surfactant and an accelerator together to form a suspension, adding particles of an inorganic salt of phosphoric acid to the suspension, and homogenising the mixture to form a paste of the ceramic based material.

In an embodiment the composite biocompatible ceramic based material is an ink for three-dimensional printing, and may be referred to as a “ceramic ink” or “bone ink” or “bio-ink”. In one embodiment, the particles of an inorganic salt of phosphoric acid is α-TCP wherein the α-TCP powder may be synthesised by mixing calcium carbonate and calcium hydrogen phosphate in a zirconia crucible at a 1 :2 molar ratio. The crucible may be placed into a high-temperature elevator furnace and the temperature increased to about 1400 °C at a heating rate of about 5 °C/min and holding time of about 5 hours. At about 1400 °C, samples are quickly removed from the furnace and quenched to room temperature. The obtained agglomerates may then be manually crushed by a mortar and pestle to achieve coarse powder with of particle size of less than about 200 μm using stainless steel sieves. The α-TCP powder may be further ground using a planetary ball mill equipped with zirconia jars and grinding balls in ethanol (weight ratio of ball: powder = 8, ethanol: powder = 3, size of zirconia balls = 3mm (2 hours, at 180 rpm) and 1 mm (2 hours, at 180 rpm) to obtain fine α-TCP powder with a narrow size distribution (D₁₀ = 1.34 μm, D₅₀ = 2.86 μm and D90 = 6.19 μm).

In one embodiment, to fabricate the ink, at the first stage the oil phase, surfactant and accelerator salt may be mixed together as, 5 wt.% (NH₄)₂HPO₄ 28 wt.% glycerol (C₃H₈O₃) and 6.5 wt.% polyoxyethylenesorbitan monooleate (C₃₂H₆₀O₁₀) in a zirconia jar for 5 minutes at 200 rpm. In a second stage, α-TCP powder (60.5 wt.%) may be added to the jar and homogenised with the suspension of precursors for 30 minutes to form a paste. Using a spatula, the paste may be mixed manually and then homogenised further using the planetary ball mill for 30 minutes. At this point, the paste may be either transferred to a syringe for printing or kept in a freezer (for example at -20 °C). Glycerol can act as the carrier for α-TCP particles which can facilitate extrusion of the bio- ink particles from an appropriate printing nozzle

In another aspect of the present disclosure is a method of fabrication of composite biocompatible ceramic material structure using an additive manufacturing process, as depicted in Figure 2, the method 100 comprises: depositing a composite biocompatible ceramic-based material 103 in a yield- stress support matrix bath 101 comprising a biological material (and/or a bioactive material), performing omnidirectional 3 dimensional printing of a structure composed of the biocompatible ceramic material integrated with the biological material, wherein the yield-stress support matrix holds the printed ceramic material 105 in place to obtain free-form omnidirectional printing of the structures.

In an embodiment wherein the yield-stress support matrix is a suspension of gelatin microspheres. The disclosure 100 in Figure 2, depicts free-form three- dimensional ceramic printing of bone-mimetic constructs with precise control of composition, without harsh chemicals or post-processing steps. Figure 2 schematically demonstrates printing free-form ceramic inks in a suspension of microspheres 102 with properties of a yield-stress fluid.

In an embodiment the method utilises a chemically stabilised gelatin microsphere support bath, where the optimised yield-stress properties can support the omnidirectional printing of a calcium phosphate-based ink in the presence of live cells as disclosed in Figure 3. The method is termed as ceramic omnidirectional bioprinting in cell- suspensions (COB ICS).

COBICS is enabled through the combination of the composite biocompatible ceramic-based material designed to use as a ceramic ink with, in some instances, a polymer microsphere suspension (or alternative support), where yield-stress characteristics support the printed ceramic structure, and nanoprecipitation “locks” the structure in place as depicted in Figure 1. The ceramic ink, which may be referred to as “bone-ink”, may be a calcium phosphate-based formulation that quickly solidifies in aqueous medium. Disclosure 300 in Figure 3, shows ceramic bioprinting 304 in the presence of live cells 301, wherein, microspheres 302 were hydrated in cell culture media 303, allowing the formation of a homogeneous bath. Here, the microspheres pack together and enter a “jammed” state where they behave like a solid under equilibrium conditions but flow like a liquid under shear forces. In this way, high-fidelity deposition of bone ink 306 occurs as the mobile printing needle 305 locally fluidizes the suspension. Once the needle front progresses, the bath destabilises and locks the printed ink in place. In an embodiment, α-TCP particles present in the composite biocompatible ceramic- based material designed to be used a as an ink in an additive manufacturing process are homogeneously dispersed in glycerol, containing ammonium phosphate dibasic and the surfactant polyoxyethylenesorbitan monooleate. Glycerol 101 acts as the carrier for a- TCP particles which may facilitates extrusion of the bone ink particles from printing nozzles. Upon extrusion of the bone-ink into aqueous environments, glycerol 101 is replaced with water 103 and hydrolysis of the α-TCP 102 particles occurs at the interface as depicted in Figure 1. During hydrolysis, ammonium phosphate may increase the pH and concentration of PO₄ ^3- in the microenvironment, which may facilitate nucleation and growth of calcium-deficient hydroxyapatite (HA) nanocrystals and ultimately quick setting of the bone-ink (Figure 1). The non-ionic surfactant may mitigate the large crack formation by modulating the growth rate of nanocrystals and ensuring a uniform crystal growth. This modulation may occur by interaction of hydrate calcium ions located on the surface of HA crystals with the oxyethylene groups of a non-ionic surfactant to form hydrogen bonds (CaOH . O(CH₂CH₂)₂). This formulation of bone-ink may facilitates fast in situ solidification through nanocrystalization in aqueous environments, converting the inorganic ink to mechanically interlocked bone apatite nanocrystals 104 (Figure 1 and Figure 7). In an embodiment the present chemically non-invasive formulation of the bone- ink enables the homogenous volumetric incorporation of bioactive molecules at high concentrations within the printed scaffold. A model protein, fluorescein isothiocyanate labelled bovine serum albumin (FITC-BSA), was incorporated in the ink prior to printing. Protein distribution was compared to the common approach of immersing sintered HA scaffolds in the protein solution.

EXAMPLES

The present disclosure will now be described with reference to the following non- limiting examples and with reference to the accompanying Figures.

Ceramic Ink Fabrication and characterisation Fabrication

Glycerol ( (C₃H₈O,₃) product number: G9012), polyoxyethylenesorbitan monooleate ( (C₃₂H₆₀O₁₀, ) product number P6224), ammonium phosphate dibasic (NaHPO₄, product number A5764), calcium hydrogen phosphate (CaHPO₄, product number C7263) and calcium carbonate (CaCO₃, product 310034) were all purchased from Sigma-Aldrich. α-TCP powder was synthesised by mixing calcium carbonate and calcium hydrogen phosphate in a zirconia crucible at a 1:2 M ratio. The crucible was placed into a high-temperature elevator furnace and the temperature increased to 1400 °C at a heating rate of 5 °C/min and holding time of 5 hours. At 1400 °C, samples were quickly removed from the furnace and quenched to room temperature. The obtained agglomerates were manually crushed by a mortar and pestle to achieve a coarse powder with of particle size of less than 200 μm using stainless steel sieves. The α-TCP powder was further ground using a planetary ball mill equipped with zirconia jars and grinding balls in ethanol (weight ratio of ball: powder = 8, ethanol: powder = 3, size of zirconia balls = 3 mm (2 hours, at 180 rpm) and 1 mm (2 hours, at 180 rpm) to obtain fine α-TCP powder with a narrow size distribution (D10 = 1.34 μm, D50 = 2.86 μm and D90 = 6.19 μm).

To fabricate the ink, the oil phase, surfactant and accelerator salt were first mixed together. In an exemplary procedure, 5 wt.% NaHPCri, 28 wt.% glycerol and 6.5 wt.% polyoxyethylenesorbitan monooleate were mixed in a zirconia jar for 5 min at 200 rpm. The α-TCP powder (60.5 wt.%) was then added to the jars and homogenised with the suspension of precursors for 30 min to form a paste. Using a spatula, the paste was mixed manually and then homogenised further using the planetary ball mill for 30 minutes. At this point, paste either transferred to a syringe for printing or kept in a freezer (-20 °C).

Characterisation of microstructure and chemistry of the designed ink material after setting

Scanning electron microscopy (FEI Nova NanoSEM 450 FE-SEM) and X-ray diffraction (The PANalytical Xpert Multipurpose X-ray Diffraction System) were used to study the microstructure and chemistry of the ink after the setting. The kinetics of the setting reaction and the associated time-dependent changes in viscous and elastic behaviour were characterised using an Anton Paar MCR 302 rotational rheometer with a 25 mm stainless steel parallel plate configuration. The ink was transferred between the plates under a dry and humid condition at 37 °C and then subjected to time-dependent oscillatory shear at a frequency of 1 rad/s and a strain amplitude of 0.04.

The ink was characterised by the following properties: injectability, cohesion, printability and setting time (Figure 6). The injectability was defined as the full extrusion of ink from a 1 mL Terumo syringe equipped with a stainless-steel tip (with an internal diameter of 600 μm). The printability was defined as the dimensional integrity of ink filaments after extrusion from a nozzle. Cohesion was defined as the physical stability of ink filaments extruded into PBS (pH 7.4) using nozzles with diameter of 220 μm, 600 μm and 1 mm. The setting time was defined as the duration after injection when the extruded filaments in PBS with 1 mm in diameter did not deform whilst being pushed by a spatula and the whole filament translocates alongside the spatula travelling direction.

For ceramic ink disks, cuboid shaped scaffolds were prepared by filling moulds of 10 mm (length) x 10 mm (width) X 3 mm (depth) with the ink and were left to set overnight at 37 °C and 5% CO₂, before being used to culture cells.

Figure 6 shows SEM images showing α-TCP particles (top), the onset of hydrolysis and degradation of α-TCP particles in contact with the aqueous environment (middle) and formation of calcium-deficient hydroxyapatite crystals (bottom).

The bone-ink takes advantage of the setting mechanism of pre-mixed calcium phosphate cements in aqueous solutions. Pre-mixed calcium phosphate cements typically consist of a mixture of α-TCP powder as the main component and a non-aqueous carrier to prevent its setting during storage. However, the pre-mixed cements suffer from drastically delayed onset and setting times, as well as containing carrier phases and/or other additives that are toxic to cells. To identify the optimum weight fraction of bone-ink components to ensure a reliable printed construct, the ink components were mixed at a variety of ratios and qualitatively assessed against four selection criteria: (i) inj ectability, (ii) cohesion, (iii) setting time and (iv) printability (Figure 8). Glycerol was noted to have a significant effect on the inj ectability, printability and cohesion, in that increasing the glycerol improved the inj ectability, but adversely affected the printability and cohesion of the ink. Excess glycerol (>35 wt.%) resulted in the merging of printed filaments (Figure 8, image A), and a low amount of glycerol (<23 wt.%) caused blockage of nozzles. Addition of ammonium phosphate substantially shortened the setting time from 30 minutes to 3 minutes, but with inferior inj ectability and post-setting crack formation (Figure 8, images B to D). It was speculated that ammonium phosphate induces non-uniform excessive growth of crystals that disallows formation of the entangled network. Increasing surfactant concentration attenuates the non-uniform crystal growth; however, at >5.0 wt.% setting time is retarded (Figure 6). One set of viable concentrations was found to be 65.8 wt.% α-TCP, 26.1 wt.% glycerol, 4.7 wt.% polyoxyethylenesorbitan monooleate and 3.3 wt.% ammonium phosphate, led to robust printing and setting.

Figure 8 depicts photographs of typical defects in ink filaments after extrusion at room temperature in the following exemplified scenarios:

Image A - merging filaments when glycerol was present in excess;

Image B - the disintegration of filaments;

Images C and D - the presence of cracks and

Image E - the leaching of components of the ink.

Figure 8, image F shows bone-ink filaments with different diameters after extrusion.

In Figure 8, image G represents a colour map of the influence of ink components in its inj ectability, cohesion, setting time and printability. The data is presented visually derived from 70 single experiments:

Inj ectability - the highlighted “red” region indicates a complete blockage of the tip or applying >200 N force for ink extrusion, the “green” region indicates complete extrusion of ink using <10 N injection force and the intermediate “yellow” region indicates complete extrusion of ink using an injection force between 10 and 100 N. Printability - the “red” area indicates a region where extruded filaments merge together, the “green” area indicates a region at which extruded filaments form a distinct interface at their intersection with no sign of merging or deformation before setting. Cohesion - the “red” region defines a condition that filaments expand substantially, disintegrate or form a large crack that leads to filament fragmentation and the “green” region defines the condition that filament expansion is minimal, filaments do not disintegrate nor form visible macro cracks. In one example, for ease of analysis, bone-ink was printed in cell culture media alone. Gelatin microspheres have been used for cell culture, either for preparation of viable cell aggregates or for controlling the fate of encapsulated stem cells. Scanning electron microscopy analysis (SEM) reveals a porous microstructure consisting of an entangled network of uniformly sized nanocrystals that resembles bone’s inorganic matrix as depicted in Figure 5. In Figure 5, representative image of direct extrusion of the bone ink in culture media are shown (pore size: -500 μm and 1500 μm (X-Y plane) and 250 μm (Z plane), and 100% interconnectivity between the pores. SEM micrographs demonstrate the nanostructured interface.

The ink setting can occur upon immediate contact with aqueous media. As shown in Figure 6, the initial set occurred in less than about 1 minute, reaching its maximum rate after about 5 minutes, with complete setting by about 10 minutes post-printing. Despite a quick setting time, and hardening in humid conditions, the bone-ink remained flowable when present in dry conditions and it showed a decreasing trend in storage modulus under rotational shear forces overtime. Under humid conditions, the complex viscosity and storage modulus of the ink increased by orders of magnitude compared to dry conditions as shown in Figure 6.

FITC-BSA distribution

Experimental 2 g of ink was directly mixed with 100 pL of fluorescein isothiocyanate labelled bovine serum albumin FITC-BSA (100 μg/mL) and then loaded into a 1 mL syringe. The syringe was loaded into the printer (Hyrel3D with a custom-designed extrusion printing head) and scaffolds were printed at room temperature into a petri-dish (8 mm x 8 mm x 2 mm in size with filament orientation = 90°). Sintered hydroxyapatite scaffolds were made by the robocasting technique and used as the control group. HA Scaffolds (5 mm x 5 mm x 5 mm in size with filament orientation = 90°) were washed with ethanol and dried at 120 °C for 2 days. They were then submerged into FITC-BSA solution (100 μg/mL) for 3 hours and washed several times with PBS. Both scaffolds types had 600 mm strut size and their cross-sections were analysed by epifluorescence microscopy (BX53F Olympus).

Results

In contrast to conventional bioceramic processing, the chemically non-invasive formulation of the bone-ink enables the homogenous volumetric incorporation of bioactive molecules at high concentrations within the printed scaffold. A model protein, FITC-BSA, was incorporated in the ink prior to printing. Protein distribution was compared to the common approach of immersing sintered HA scaffolds in the protein solution. Fluorescence characterization indicates BSA is spread throughout the bone-ink filament, in contrast to perimeter localization in sintered scaffolds as depicted in Figure 9.

Drug release

Experimental

Dexamethasone and ibuprofen, two commonly used drug models for assessing encapsulation and release, were incorporated into the bone-ink prior to printing. Dexamethasone (D4902, Sigma-Aldrich) and ibuprofen (14883, Sigma-Aldrich) were selected as the model drugs. The dexamethasone and ibuprofen were mixed in powder form with the ink to obtain scaffolds containing 1 mg of the drug. For the control group, sintered HA scaffolds were submerged into drug solution (lmg/mL in ethanol) for 3 hours. Drug loaded scaffolds were washed with PBS several times and placed in airtight containers in PBS at 37 °C in a shaker incubator. At each time point, 3 mL of PBS was removed to measure the amount of the released drug and 3 mL fresh PBS was added to each container.

Results Figure 10, image A depicts comparative drug release profiles of dexamethasone and ibuprofen loaded into bone-ink scaffolds and sintered hydroxyapatite scaffolds showing higher controlled release when drugs are incorporated in ink.

In contrast to immediate release of physiosorbed drug from sintered scaffolds, which resulted in no detection of drug after the first minute, the bone-ink showed sustained drug release profiles with 60% by day 3 and 100% by day 15 (Figure 10, image

A).

For FITC-BSA, ibuprofen, and dexamethasone, the total mass of the molecule in respect to the mass of the bone-ink for each scaffold was 0.0005, 0.5 and 0.5 wt%, respectively. The ink stability may be sensitive to excessive addition of aqueous or organic phases within which biomolecules are dissolved since wet-phases alter the rheology attributes, integrity and kinetics of crystallization in the ink. Therefore, at concentrations which biomolecules are therapeutically effective (substantially lower than the mass of ink), size, charge and chemistry of the biomolecules may not dictate a significant change in ink properties.

The compressive strength of printed scaffolds was measured at a range of porosities (30 to 85%) by changing the pore size, after being kept in PBS for one week at 37 °C. The compressive strength of the constructs was in the range of strength of cancellous bone and higher than that for sintered scaffolds (Figure 10, image B).

Computational modelling Experimental

Gelatin microspheres were modelled in five different sizes, with diameters 600 μm, 500 μm, 400 μm, 300 μm, and 20 μm. With respect to geometrical variables, ink bars of two sizes (600 mm and 200 mm in diameter), two geometrical shapes (straight and spiral shapes) and three spatial orientations (including vertical, 45° inclination and horizontal groups), were considered in this study. Figure 11 A, images B to D show representative assemblies of the container, gelatin microspheres (600 μm and 300 μm in diameter) and ink bar. The container and ink bar were the same in both conditions, and the ink bar was placed in the middle of the container. The gelatin microspheres were uniformly distributed throughout the rest of the container. More specifically, the gelatin microspheres were arranged layer by layer, to be staggered in both vertical and centripetal directions, shown by the top view and transparent 3D view. The potential configuration of ink bars in the current study are as follows: straight bar with 600 μm diameter, straight bar with 200 μm diameter, and spiral bar with 200 μm diameter respectively. The spatial orientation of the ink bar for each group includes the vertical, 45° inclined, and horizontal direction. Both mechanical and diffusional properties used in the present study are summarized in Table 1. Table 1 - Material properties for each component in the system

It should be noted that the oxygen diffusivity and solubility coefficient were measured at the specific working conditions when the temperature was 37 °C and standard atmospheric pressure. All of the gelatin microspheres, ink bar and water were meshed by using linear tetrahedral elements. A sensitivity analysis of the mesh was conducted to determine the appropriate global mesh size, which was found to be 30 μm for all assembled models. This step is used for avoiding excessive computational costs, as well as ensuring the adequate accuracy of the simulation results. The gelatin microspheres with 600 μm diameter were meshed with 440,696 linear tetrahedral elements (degree of freedom - DOF : 278,703), 500 μm group were meshed with 598,606 linear tetrahedral elements (degree of freedom - DOF: 412,698), 400 μm group were meshed with 775,780 linear tetrahedral elements (degree of freedom - DOF: 538,336), and 300 μm group were meshed with 945,720 linear tetrahedral elements (degree of freedom - DOF: 629,685). The ink bar with 600 μm diameter was meshed by 19,215 linear tetrahedral elements (degree of freedom - DOF: 11,859), and 200 μm diameter group was meshed by 2,215 linear tetrahedral elements (degree of freedom - DOF: 1,467). In addition, the spiral ink bar was meshed by 37,244 linear tetrahedral elements (degree of freedom - DOF: 24,093). Dynamic/Explicit module in Abaqus 2016 (ABAQUS, Inc, Providence, RI) was adopted to perform the traditional deformation finite element analysis, since the simulation in this study is highly nonlinear, and includes the interaction between solids and fluids. The system was only under the load owing to gravity, and the gravitational acceleration (g) was set as 9.8 m/s². A boundary condition was applied on the bottom surface of container, by kinematically constraining the vertical displacement. For the diffusion analysis, mass diffusion module was adopted to conduct the diffusion analysis, and the surface concentration flux load was created to generate the gradient concentration of oxygen from the free surface (top surface) to the middle part of container and all the way to the bottom. The initial inlet concentration was set as 20 (% or mmol/cm³) and uniformly distributed around the top surface.

Results

Towards an optimal yield-stress matrix capable of free-form bioceramic printing, computational studies were first performed to determine the interrelationship between bone-ink filament diameter and microspheres size to obtain the highest printing resolution. By analysing combinations of microspheres, spatial distribution, and various configurations of bone-ink filaments, either horizontal, vertical, or with a 45° inclination (Figure 11, image A), it was found that the largest ink deformation was more likely to be concentrated in the bottom region of filaments, regardless of size and geometry (Figure 11, images B to D). Moreover, thinner filaments led to larger magnitudes of deformation (Figure 11, images B and C). When comparing ink with similar diameter, the spiral geometry further increased peak deformation values compared to straight filaments (Figure 11, images C and D). Decreasing filament diameter resulted in less indented deformation, especially in the vertical and 45° inclination placements. When analysing the deformation distribution for the linear filaments of the same diameter supported in different size microsphere suspensions, it was found that the peak deformation values increased with microsphere size. With respect to the spatial orientation of the filaments, the deformation decreased when changing the orientation from vertical to horizontal (Figure 11, image B).

Figure 11, image A provides a representative assembly of gelatin microspheres and ink filaments; images B to D present the deformation results for the filaments.

Figure 11, image B provides the modelling data for the ink filaments with 600 μm diameter and under different loading conditions owing to the change in sizes of microsphere, including 600 μm, 300 μm and 20 μm, and filament orientation, including vertical, 45° inclination and horizontal groups.

Figure 11, image C provides the modelling of filaments with 200 μm diameter and image D the spiral ink filament with 200 μm in diameter.

Figure 11, images E to G present the oxygen concentration, particularly: image E - oxygen in the nutrient solution; image F - oxygen in the gelatin suspension, and image G - oxygen in the bottom layer of gelatin suspensions. The diffusion time period was set as 5 hours and the concentration (%) contour of water is shown in images E to G. The difference between the maximum and minimum oxygen concentration in nutrient solution was less than 1% and it indicated that the quasi-steady state of oxygen was achieved. The gradient oxygen concentration, decreasing from top to bottom, can be observed in both solution medium and gelatin microspheres medium (image E) and (image F). The bottom layer of the gelatin microspheres was selected as the representative layer to show more details of concentration distribution in gelatin microspheres (image G). The lower oxygen concentration was found in the middle region, caused by the solid ink bar blocked the oxygen flow from the top surface. Regarding the outer layer of spheres, the higher oxygen concentration was found in the marginal region. Since the effect of top surface flux load was compromised, the diffusion effect from surrounding water medium to gelatin microsphere becomes more prominent, owing to the difference in oxygen concentration.

Gelatin microspheres fabrication and characterisation

Experimental

Gelatin type A (G2500 Sigma) was dissolved in deionised water at 50 °C before sterile filtration. For size M particles and size L, an oil bath (Canola Oil) was pre-warmed to 40 °C under slow and vigorous stirring respectively. A 2.66% (v/v) gelatin solution was added dropwise and allowed 10 minutes to stabilise in the emulsion. The bath was subsequently cooled down to 10 °C and held for 30 minutes before the addition of 20% (v/v) solution of 0.25 % (w/t) glutaraldehyde in acetone (Sigma). The particles were allowed 4 hours to dehydrate and crosslink before decanting the mixture into centrifuge tubes to be extensively washed with acetone. The microparticles were then sonicated for 30 seconds before being stored in an acetone solution until further use. For the size S particles, 1.5 %(w/t) of span 80 (Sigma) was added to the oil bath prior to the addition of gelatin to stabilise the emulsion. The bath temperature was also pre-warmed to 55 °C at the start. For the control samples, 4 hours of mixing with the glutaraldehy de-acetone solution was replaced with 1 hour of mixing with pure acetone.

Results

Based on the modelling results, gelatin microspheres were fabricated with diameters of 14.0 ± 6.31 μm (S), 93.3 ± 45.8 μm (M) or 401μm ± 187 μm (L) (Figure 12) to evaluate printing fidelity as a function of microsphere size. Medium-sized gelatin microspheres (M) were synthesised using a standard water-in-oil emulsion at 40 °C with rigorous rotation to form droplets, followed by cooling to 10°C to physically crosslink the microspheres. Reducing the rotational speed of the oil bath resulted in larger microspheres (L); while increased rotational speed paired with emulsion-stabilising surfactant (Span 80) created smaller microspheres (S). The fabricated gelatin microspheres would dissolve at 37 °C so that printed structures could be harvested directly. However, for some applications, a stable suspension may be advantageous, e.g., for using the gelatin as a complex microstructured extracellular matrix.

To stabilise the suspension, glutaraldehyde was added to the freshly made microspheres with stirring. The simplicity of the system will allow other crosslinking schemes including mild approaches using enzymes, thereby tuning the susceptibility to degradation in biological environments.

Figure 12 shows the size distribution and optical image of crosslinked gelatin microspheres by glutaraldehyde at fully hydrated state. Image A shows the optical image (20x ) of hydrated microparticles synthesised with span surfactant (S) and histogram of size distribution. Image B shows the Optical image (·4x) of hydrated microparticles synthesised under standard conditions (M) with the histogram of size distribution. Image C shows the optical image (4x) of hydrated microparticles synthesised under slow conditions (L) with the histogram of size distribution. Scale bars: (A) 50 μm, (B and C) 350 μm.

Cell culture Experimental

Human osteoblasts (hOF) and human bone marrow derived stem cells (BM-MSC) were used to test ink biocompatibility. hOF were purchased from ATCC and cultured in a 1 : 1 mixture of Ham's F12 Medium Dulbecco's Modified Eagle's Medium, with 2.5 mM L-glutamine (Gibco) supplemented with 10% fetal bovine serum (Bovogen) and 1% penicillin/streptomycin (Invitrogen), to which hereon we will refer as osteoblast media. BM-MSC were acquired by Lonza, cultured, and expanded in fully supplemented mesenchymal stem cell basal medium (Lonza) according to the manufacturer’s instructions. Human adipose derived mesenchymal stem cells (ADSC) were obtained from ATCC, cultured and expanded in fully supplemented Mesenchymal Stem Cell Basal Medium for Adipose, Umbilical and Bone Marrow derived MSC (ATCC). All cells were then cryopreserved in 10% dimethyl sulfoxide solution at passage 2. Both BM- MSC and ADSC were subsequently thawed and cultured in the so-called complete medium, containing Dulbecco’s modified Eagle’s medium and low glucose (1000 mg L^-1) (DMEM, Invitrogen) media supplemented with 10% fetal bovine serum (Bovogen) and 1% penicillin/streptomycin (Invitrogen). hOF were thawed and cultured in the osteoblast media.

Medium was changed every 3 days, and cells were passaged at 70% confluency using a solution containing 0.25% trypsin/1 mM. Cells were then used for experiments between passage 4 and 9. When requested, ADSC were switched to osteogenic culture media, consisting of DMEM supplemented with 100 nM dexamethasone, 10 mM b- glycerol phosphate, and 0.05 mM ascorbic acid (all from Sigma-Aldrich). Optimisation experiments for gelatin microsphere and cell concentration

Figure 13 shows optimisation experiments for gelatin microsphere and cell concentrations. Adipose-derived mesenchymal stem cells (ADSC) were cultured either at concentration 1 (Cl), corresponding to 2x 10⁴ cells per sample (image A) or at concentration 2 (C2) of 4x 10⁴ per sample (image B). G1= centrifuged microspheres hydrated in a small volume of culture media, G2= microspheres first mixed with cells and then centrifuged to remove cultured media; L= microspheres hydrated with culture media and thus mixed with cells in the liquid phase. Scale bars = 100 μm.

Figure 14 depicts cell-microsphere size compatibility. Image A shows ADSC cultured with different sizes of gelatin microspheres: S, M and L. Cell viability was assessed after 7 days by live (green) /dead (red) fluorescence staining. Image B show's 3D reconstruction of live/dead images used for quantifying cell density in each group. Image C whose cell/microspheres ratio in S, M and L conditions. Image D shows ADSC cultured at low (1x10⁴ cells/mg microspheres) and high (5x10⁴ cells/mg microspheres) concentration, either in the presence of S or M size spheres. Cell viability was tested at day 1 (left) and day 14 (right). Image E is a table showing cell density in each condition represented in d. Scale bars 100 μm.

COBICS printing process

A multi -head printer (Engine HR, Hyrel 3D, USA) equipped with a customized extruder (EMO-25) for COBICS was used. For ink extrusion, 1 mL of ink was loaded into a 3 mL printing syringe and syringe was inserted in the EMO-25 extruder. All needles used in the study were purchased from Nordson EFD with inner diameter of 0.2 to 0.8 mm. The compressive strength of scaffolds was measured using uniaxial compressive tests utilizing a universal testing machine equipped with a 5 kN load cell at a constant crosshead speed of 1 mm.min^-1. Five specimens for each porosity (approximately 85%, 70%, 60%, 50%, 40% and 30%) was tested. The target porosity was obtained by varying spacing between the fdaments (changing the pore size). The compressive strength was calculated by dividing the maximum load at the fracture point by the cross-sectional area of the samples. The scaffolds with strut size of 0.8 mm and orientation of filaments of 0°/90° were printed at room temperature using a Geode to obtain scaffolds with 8 mm x 8 mm x 8 mm in dimension. The scaffolds were tested 7 days after incubation in PBS at 37 °C. For printing bone mimicked structures, the computer model was created by Autodesk Fusion 360 and then converted to STL file (Figure 19) prior to generating the Geode. A 0.2 mm needle was used to print the constructs in 96 wells of cell culture plates containing gelatin microspheres and culture medium. After printing, constructs were kept in supporting bath for 6 minutes in order to setting reaction completes then they were removed by a tweezer.

For the development of COBICS construct including cells, 2% agarose cylindrical shape well moulds (5 x 3 mm) (Sigma-Aldrich) were used to contain the COBICS construct. One day before printing, 6 x 10⁵ ADSC were mixed with 12 mg gelatin microspheres and deposited in each agarose mould containing complete media. On the following day, the ink was extruded in the gelatin-cell colloid bath with a 1 mL syringe and 23 gauge tips (Nordson) and incubated at 37 °C and 5% CO₂. Samples were kept up to 14 days and media was changed every second day.

Gelatin microspheres have been used for cell culture, either for preparation of viable cell aggregates or for controlling the fate of encapsulated stem cells. Therefore, it is reasoned that addition of cells to the gelatin bath would facilitate adhesion and proliferation. For this purpose, adipose-derived mesenchymal stem cells (ADSC) were incorporated in the bath phase, by mixing cells with gelatin microspheres for 1 day, followed by printing with the bone-ink (Figure 3). Two different cell concentrations were tested, 5x10³ and DIO⁴ cells per mg of 10 microspheres. Cell numbers were chosen based on previous studies of ADSC cultured in 3D matrices. All groups show high cell viability when tested up to day 7, with higher cell concentration corresponding to higher viability (Figure 13). cell integration with different sized microsphere suspensions was evaluated. Across all groups, no dead cells or any sign of overt toxicity were observed (Figure 14, image A). Cells showed the highest density when cultured with the 15 largest microspheres, corresponding to 1500% after 14 days of culture, with evidence for robust adhesion, spreading and proliferation (Figure 14, images B and C). Increasing cell number to 5x10⁴ cells/mg of microspheres in either S or M suspensions increased cell viability and density (Figure 14, images D and E). Since printing fidelity decreases as microsphere size increases, M suspensions were selected for the remaining printing experiments.

Having established that cells could survive and proliferate within the microsphere suspension, the biocompatibility of cells directly adherent to the bone-ink material was explored. Mesenchymal stem cells (MSCs) and bone-specific osteoblasts were cultured directly on rectangular-shaped scaffolds entirely composed of bone-ink for up to 7 days. MSCs adherent to the ceramic scaffold showed high viability (100%) after 24 and 72 h (Figure 4, image b left and Figure 4, image B, i). Moreover, the MSCs acquired an elongated shape, confirmed by cell aspect ratio quantification, when cultured for 3 days directly on the bone-ink, compared to cells seeded on tissue culture plates (Figure 4, image D, ii). After 7 days of culture, MSCs formed a dense cell sheet, with bone-like 5 nodules arising from colony-forming units, which is suggestive of osteogenic differentiation, as shown previously using in vitro bone models with MSCs.

Since cells are compatible with the gelatin microspheres, bone-ink and composite material, the potential to fabricate a suspended free-form construct in the presence of living cells was next investigated. A bath of gelatin microspheres was loaded with ADSC for printing three dimensional bone-ink structures. Within hours, uniform cell integration was observed within the support matrix. After 14 days, cells appeared to proliferate within the gelatin matrix as well as along the gel-ink interface (Figure 4, image C) and were over 95% viable (Figure 4, image D, right). On day 7, a slight decrease in cell viability at the bone interface was noted (Figure 4, image D, right), suggesting the onset of osteogenic differentiation. Live/dead staining demonstrates high viability at day 1 (97.71 ± 3.24 %), day 7 (82.83 ± 13.41 %) and day 14 (91.95 ± 4.23 %).

Figure 3 depicts COBICS showing a schematic of ceramic bioprinting in the presence of live cells. Figure 4 image B provides a top view of a three dimensional printed construct after 7 days 5 culture with mesenchymal stem cells (MSCs), showing tissue formation around the ink and live- dead images of day 14 cell culture in COBICS. Figure 4 image C shows the viability staining of MSCs and osteoblast cells seeded on bone-ink and scanning electron microscopy of MSCs on bone-ink after 7 days, showing the production of mineral nodules indicating the transition of MSCs toward osteogenic lineage. Figure 4 image D shows cell viability and cell shape analysis of MSCs cultured on ceramic ink, cell viability of human osteoblasts on ink, , MSCs viability embedded within the support bath and migration of MSCs toward bone-ink. To evaluate the osteoinductive potential of COBICS structures, gene and protein analysis of early, intermediate and late-stage markers of osteogenesis in MSCs were performed. After 1 week of culture, RT-PCR showed a 20-fold increase in the early marker runx-2, compared to cells cultured on tissue culture plates and a 2-fold increase compared to cells cultured on tissue culture 20 plates with osteogenic induction media (Figure 18, image A). Runx-2 is a master transcription factor associated with osteogenesis, as it has been shown to upregulate osteoblast-related genes, such as collagen type I, bone sialoprotein (BSP), osteocalcin and alkaline phosphatase. Furthermore, markers for later phases of osteogenesis, including BSP (Figure 18, images B), osteocalcin (Figure 18, image C) and osteopontin (Figure 18, image D)were similarly up-regulated compared to cells cultured on standard plates.

Figure 18 depicts differentiation of mesenchymal stem cells in 3D printed composites. Images A to D relate to osteogenic gene expression: Runx-2, bone sialoprotein (BSP), osteocalcin and osteopontin, for cells cultured in COBICS composites relative to tissue culture plates. Schematic representation of cells present in gelatin bath, far from the ink (image E) and in proximity to the bone-ink (image F). Representative images of cells stained for osteopontin (red in true image) in the vicinity of the bone-ink (images F, H and J) and far from the bone-ink (images E, G and I), either in expansion (images G and H) or osteogenic medium (images I and J). Nuclei were counterstained with DAPI (blue in true image). Scale bars = 200 μm.

Markers for later phases of osteogenesis, including BSP (Figure 18, image B), osteocalcin (Figure 18, image C) and osteopontin (Figure 18, image D) were similarly up-regulated compared to cells cultured on standard plates. Remarkably, cells cultured on COBICS showed a further increase in expression compared to cells cultured in the presence of biochemical factors commonly used for inducing in vitro osteogenic differentiation. Other ceramic biomaterials, such as HA scaffolds, have shown two to three fold increased expression of early markers (runx-2). However, only osteocalcin showed a similar increase compared to COBICS. These results suggest that the disclosed bone-ink is a highly osteoinductive material. Osteopontin expression was additionally confirmed by protein analysis, both in expansion media (Figures 18, images E and F) and when supplemented with osteogenic factors (Figure 18, images G and H). Under growth conditions, cells expressed osteopontin only in close proximity to the bone-ink and when adherent to the ink itself (Figure 18, image H), whereas cells far from the ink did not show positive expression (Figure 18, image G). When cultured in osteogenic media cells express osteopontin throughout the multiphasic construct (Figures 18, images I and J). Irregular bones function as protectors of internal organs, and as such they possess complex structures that are difficult to be artificially reproduced. To evaluate the use of COBICS for fabrication of irregular bone structures, mimics of the human osseous labyrinth (Figure 16, image D), trabecular bone (Figure 16, image E) and haversian canals (Figure 16, image F) were printed. In these cases, a suspension of unmodified gelatin microspheres was used, so that after fabrication the printed bone is recovered through gelatin dissolution at 37 °C (Figure 19). The potential for COBICS to recreate multiphasic interfaces was also investigated. The human body presents four main categories of interfaces, namely cartilage-bone, ligament-bone, tendon-bone and muscle- tendon. A multiphasic construct, with bone represented by the ceramic ink, and soft- tissue represented by the surrounding microsphere matrix (Figure 16, image C) were recreated. The versatility of the emulsion approach will allow incorporation of virtually any matrix protein within the microspheres, and integration with tissue-specific cells will further expand the capabilities for mimicking complex tissue interfaces.

Figure 16 shows fabrication of complex bone-mimetic architectures. Image A includes photographs of a printed bone-5 ink filament within a gelatin microsphere support bath and interfacial adhesion during nanocrystal formation. Image B is a front view of the printing process in a 96-well cell culture plate. Image C is an example of multiphase construct for potential application in osteochondral defect; generation of high-resolution bone mimicked constructs with hierarchical microstructure, such as human osseous labyrinth (image D), trabecular bone (image E) and helical haversian canal (image F).

Figure 19 provides examples of computer-generated three-dimensional models (top) and corresponding 3D printed bone-like structures (bottom) through COBICS technique.

Having established optimal bone-ink formulation and setting parameters, next is printing in the presence of biological materials investigated. Since most bones present highly interconnected complex structures, it was reasoned that optimizing a yield-stress support matrix to hold a printed ceramic material in place would enable free-form printing of complex ceramic shapes. Rheological analysis of gelatin microspheres bath Experimental

All rheological measurements were performed on an Anton Paar MCR 302 Rheometer with parallel plate geometry (25 mm disk, 1 mm measuring distance) at 25 °C. The gelatin microsphere bath was prepared the same way as for printing experiments, and 600 μl of bath was added to the instrument stage. Oscillatory measurements were taken with a 1 Hz frequency with a log ramp of shear strain rate from 0.05%/s to 100%/s over the course of 10 minutes.

Results Figure 15 depicts the rheology of glutaraldehyde treated gelatin microsphere bath.

Image A shows complex viscosity versus shear strain rate and, image B shows complex viscosity versus shear stress. Shear rheology confirmed that the gelatin microsphere suspensions behave as yield-stress fluids — responding as a rigid body under low shear stress, whilst as a viscous fluid under high shear stress as shown in Figure 15.

Live dead assay Experimental

A live/dead assay was performed to both assess viability of cells directly cultured on ink-based disks and or printed by the COBICS system. For this purpose, cell viability was analysed after up to 14 days culture. To briefly describe the protocol used, cultured media was removed from each sample, which was then washed with Dulbecco’s phosphate-buffered saline (DPBS, Gibco). After removing DPBS, samples were incubated with a solution of DPBS containing 2 mM calcein (Thermo Fisher Scientific) and 4 mM ethidium homodimer-1 (Thermo Fisher Scientific) at 37 °C for 1 hour. Following this, staining solution was removed, samples were washed with DPBS and transferred in a glass bottom dish for imaging through Nikon A1 confocal microscope at 488 nm to detect live cells and 543 nm for dead cells. Cell viability was then quantified using Image-J software. Cell fixing, staining and confocal imaging

Cells were fixed by fixing samples in 4% paraformaldehyde (Sigma- Aldrich) at 4 °C for 24 hours. Samples were then treated with a protocol for clearing, which included a 2 day incubation at room temperature (RT) in a solution for decolourisation and delipidation containing (25 wt% urea, 25 wt% N,N,N',N'-tetrakis(2-hydroxypropyl) ethylenediamine and 15 wt% Triton X-100 (all Sigma). The samples were then washed several times in PBS, then incubated with primary antibodies against the osteogenic markers, anti-osteocalcin (1:100, Sigma-Aldrich) and anti-osteopontin (1:100, Abeam) at RT for 1 day, and then with secondary antibodies anti- mouse Alexa Fluor® 488 (1 :200; Thermo Fisher Scientific), anti-rabbit Alexa Fluor® 647 (1 :200, Thermo Fisher

Scientific) and DAPI (Sigma-Aldrich). Samples were then further incubated in CUBIC- 2, containing a mixture of 50 wt% sucrose, 25 wt% urea, 10 wt% 2,2,2'-nitrilotriethanol and 0.1% (v/v) (all Sigma) for another 2 days at RT, and then imaged immerged in same solution for matching the refractive index, by Nikon A1 confocal microscope. Images were collected using the following laser wavelength settings: DAPI using 405 nm, osteocalcin using 488 nm, and osteopontin using 645 nm.

Quantitative RT-PCR analysis

For mRNA expression analysis of ADSC cultured either in standard TCP or in COBICS system, cells were maintained in culture for the required days at 37 °C, in 5% CO₂ and 20% O₂. Subsequently, cells were collected from each sample by trypsin treatment, and standard RNA isolation protocol was performed according to the manufacturer's instructions (Qiagen). 500 ng of total RNA was reverse transcribed into cDNA with a random hexamer primer using high-capacity cDNA reverse transcription kit (Applied Biosystems) according to the manufacturer's instructions and then reverse transcription-polymerase chain reaction (RT-PCR) was performed using CFX96 real- time detection system (Biorad). The reaction mixture was composed of 10 pi of SYBR Select Master Mix (Applied Biosystems), 10 μmol each of the forward and reverse primers, 2 μl of cDNA, and distilled water to a final volume of 20 μl. The thermocycling conditions were 95 °C for 30 seconds, followed by 40 cycles of 95 °C for 5 seconds and 60 °C for 34 seconds. Normalisation of the data was performed using the housekeeping gene glyceraldehyde-3- phosphate dehydrogenase (GAPDH) as an endogenous control in the same reaction as the gene of interest. The primers used in this study are listed in Table 2. The specificity of the SYBR PCR signal was confirmed by melt curve analysis. Ct values were transformed into relative quantification data using the 2^_ΔΔCt method, and data were normalised to GAPDH mRNA expression. Table 2 - List of primers’ sequences used for evaluation of the expression of osteogenic markers.

Statistical analysis

Statistical analyses were performed using GraphPad Prism (version 8) software with 3-5 samples analysed for each experimental group. One-way analysis of variance (ANOVA) was used for analysis of variance to compare between groups. Unless defined otherwise, results are provided as mean ± standard deviation of n ≥ 3 independent experiments. Values were considered significant when p < 0.05.

Fabrication of complex bone-mimetic architectures

Free-form printing of the bone-ink in the different suspensions of gelatin microspheres was explored such that, microspheres were hydrated in cell culture media, allowing the formation of a homogeneous bath. Here, the microspheres pack together and enter a “jammed” state where they behave like a solid under equilibrium conditions but flow like a liquid under shear forces. In this way, high-fidelity deposition of ink occurs as the mobile printing needle locally fluidizes the suspension. Once the needle front progresses, the bath destabilises and locks the printed ink in place. Imaging the ink setting within the support bath demonstrates firm adhesion of the gelatin microspheres to the nanostructured bone-ink interface (Figure 16, images A and B). Microspheres in direct contact with the bone-ink remain fixed while the surrounding microspheres retain yield-stress fluid characteristics (Figures 8 and 10). When the bone-ink is deposited in the support bath, the extruded filaments do not disintegrate and keep their rigidity even at diameters as small as 200 μm (Figure 16, image A, left). This confirms the ability to accurately print delicate stmctures, which by proximity nucleates nanocrystals which mimic natural bone structures. This is clearly visualized by increased surface roughness of the ink compared to initial contact (Figure 16, right). Increasing the microsphere size resulted in deviations in extruded filament diameter compared to nozzle diameter with L>M>S as shown in Figure 17.

Figure 17 shows the effect of gelatin microsphere size on the deformation of ink filaments extruded through nozzles with diameters of 220, 430 and 500 μm. As a general trend, increasing the microsphere size resulted in a more pronounced deformation in diameter of printed filaments. The printing bath containing microspheres with an average size of 20 μm caused the least deviation in filament diameter.

In summary, the techniques disclosed herein may provide an alternative approach to traditional ceramic-based scaffold fabrication, where a formulation as described herein can induce rapid crystallization in aqueous environments, with the ability to integrate bioactive molecules. Printing the ink in a suspension of matrix and cells, with characteristics of a yield-stress fluid, can facilitate free-form omnidirectional printing, with subsequent cell engraftment and differentiation. The high-density microsphere suspension surrounding the printed architecture can promote robust cell adhesion and proliferation, potentially with greater than 95% viability after several weeks in culture. In some instances, he tendency for cells to differentiate at the interface of the bone ink, while remaining multipotent in the intervening spaces opens the potential for fabricating gradient tissue structures, and intervening vasculature. Ultimately, this approaches disclosed herein may enable the in-situ fabrication of bone-like tissues with the potential for using patient cells and in vivo imaging to guide the design (e.g. computed tomography, magnetic resonance imaging), which may have broad implications for disease modelling, drug discovery, and regenerative engineering in clinical settings.

Ceramic omnidirectional bioprinting in cell-suspensions may overcome the major challenges in printing ceramic materials for bone regeneration: (i) a cell-friendly ceramic ink printable at room temperature, with fast setting time in aqueous environments, (ii) tunable composition for inclusion of drugs and biomolecules without post-processing steps, (iii) anisotropic deposition to create complex bone-mimetic shapes, and (iv) a supporting cell-laden matrix for in situ tissue integration. The ability to print nanostructured bone-mimetic ceramics within cell-laden biological materials in free- form with control over macro- and micro-architecture, provides scope for complex bone mimicry and real-time bone reconstruction in clinical settings. EXAMPLE EMBODIMENTS

The present disclosure may be described by the following example embodiments.

1. A composite biocompatible ceramic based material comprising particles of an inorganic salt of phosphoric acid, wherein the particles of the inorganic salt of phosphoric acid are dispersed in a mixture comprising an oil phase and a surfactant.

2. The composite biocompatible ceramic based material of example embodiment 1, wherein the inorganic salt of phosphoric acid is alpha-tri calcium phosphate (a- TCP).

3. The composite biocompatible ceramic based material of example embodiment 2 wherein the α-TCP is synthesised by mixing calcium carbonate and calcium hydrogen phosphate, optionally in a 1 :2 molar ratio.

4. The composite biocompatible ceramic based material of any one of example embodiments 1 to 3, wherein the oil phase is glycerol.

5. The composite biocompatible ceramic based material of any one of example embodiments 1 to 4 wherein the surfactant is a non-ionic surfactant.

6. The composite biocompatible ceramic based material of example embodiment 5 wherein the non-ionic surfactant is polyoxyethylenesorbitan monooleate.

7. The composite biocompatible ceramic based material of any one of the preceding example embodiments further comprising an accelerator.

8. The composite biocompatible ceramic based material of example embodiment 7, wherein the accelerator is ammonium phosphate dibasic (APD).

9. The composite biocompatible ceramic based material of example embodiment 7, wherein the accelerator is sodium phosphate dibasic.

10. The composite biocompatible ceramic based material of any one of the example embodiments 1 to 9, wherein between about 50 and 70 wt.% of particles of inorganic salt of phosphoric acid is dispersed in a mixture of about 20 - 35 wt.% of the oil phase, about 5-10 wt.% of the surfactant and about 3-8 wt.% of an accelerator.

11. An ink for additive manufacturing, the ink composed of a composite biocompatible ceramic-based material which when deposited in or on a yield-stress support matrix in the presence of at least one bioactive molecule and/or biological material, and allows free-form three dimensional printing of ceramic bone mimetic shapes integrated with the at least one bioactive molecule and / biological material.

12. The ink of example embodiment 11, wherein the composite biocompatible ceramic-based material comprises particles of an inorganic salt of phosphoric acid; the particles of inorganic salt of phosphoric acid being dispersed in a mixture comprising an oil phase and a surfactant.

13. The ink of example embodiment 11 or example embodiment 12, wherein the ink composed of a material which when deposited in or on a yield stress support matrix forms free-form three-dimensional printing of simple and complex ceramic bone mimetic shapes integrated with the at least one bioactive molecule and/or biological material.

14. The ink of any one of example embodiments 11 to 13, wherein the yield stress support matrix is a suspension of gelatine microspheres.

15. The ink of any one of example embodiments 11 to 13, wherein the composite biocompatible ceramic based material is the composite biocompatible ceramic based material of any one of example embodiments 1 to 10.

16. A method of making a composite biocompatible ceramic based material, the method comprising mixing an oil phase, a surfactant and an accelerator together to form a suspension, adding particles of an inorganic salt of phosphoric acid to the suspension, and homogenising the mixture to form a paste of the ceramic based material.

17. A method of fabrication of a composite biocompatible ceramic material structure using an additive manufacturing process, the method comprising: depositing a composite biocompatible ceramic-based material in a yield- stress support matrix bath comprising at least one bioactive molecule and/or biological material, performing omnidirectional 3 dimensional printing of a structure composed of the biocompatible ceramic material integrated with the at least one bioactive molecule and/or biological material, wherein the yield-stress support matrix holds the printed ceramic material in place to obtain free-form printing of the structures.

18. The method of fabrication of a composite biocompatible ceramic material structure of example embodiment 17, wherein the composite biocompatible ceramic-based material comprises particles of an inorganic salt of phosphoric acid, the particles of inorganic salt of phosphoric acid being dispersed in a mixture of an oil phase and a surfactant

19. The method of fabrication of a composite biocompatible ceramic material structure of example embodiment 17 or example embodiment 18, wherein the yield-stress support matrix is a suspension of gelatin microspheres. 20. The method of fabrication of a composite biocompatible ceramic material structure of example embodiment 19, wherein the gelatin microspheres dissolve at about 37 °C.

21. The method of fabrication of a composite biocompatible ceramic material structure of any one of example embodiments 16 to 20, wherein the at least one biological material is live cells.

22. The method of fabrication of a composite biocompatible ceramic material structure of any one of example embodiments 16 to 21, wherein an anti-inflammatory medication is added to the biocompatible ceramic material before the step of printing.

23. The method of fabrication of a composite biocompatible ceramic material structure of any one of example embodiments 16 to 22, wherein at least one bioactive molecule is added to the biocompatible ceramic material before the step of printing.

24. The method of fabrication of a composite biocompatible ceramic material structure of example embodiment 23, wherein at least one bioactive molecule is fluorescein isothiocyanate labelled bovine serum albumin (FITC-BSA).

25. The method of fabrication of a composite biocompatible ceramic material structure of any one of example embodiments 16 to 24, wherein fabrication is performed at about room temperature.

26. The method of fabrication of a composite biocompatible ceramic material, wherein the composite biocompatible ceramic material is defined by any one of example embodiments 1 to 10.

27. A bone like structure mimicking bone and formed by the method of any one of example embodiments 16 to 26 using the composite biocompatible ceramic-based material of any one example embodiments 1 to 10 or the ink of any one of example embodiments 11 to 15.

28. The bone like structure of example embodiment 27, wherein the structure is a high resolution construct with a hierarchical microstructure.

29. The bone like structure of example embodiment 28, wherein the high-resolution construct is a multiphasic construct with hierarchical microstructure.

30. The bone like structure of embodiment 28, wherein the construct with hierarchical microstructure is an irregular bone structure mimicking one of a human osseous labyrinth, a trabecular bone or a helical haversian canal.

31. The bone like structure of any one of embodiments 27 to 30, wherein the structure is integrated with biological cells. 32. The bone like structure of any one of embodiments 27 to 31, wherein the structure incorporates at least one bioactive molecule and/or biological molecule.

33. The bone like structure of any one of embodiments 27 to 32, further comprising one or more drugs supporting sustained drug release.

34. The bone like structure of embodiment 33, wherein the drugs are selected from dexamethasone and ibuprofen.

35. The bone like structure of embodiment 32, wherein the bone like structure comprises a protein or fluorescein isothiocyanate labelled bovine serum albumin (FITC-BSA).

36. A method of using the composite biocompatible ceramic-based material of any one embodiments 1 to 10 or the ink of any one of embodiments 11 to 15 as a bone cement.

37. The method of embodiment 36, wherein the bone cement is used to bond two or pieces of bone together and/or bond one or more synthetic materials to bone.

38. A method of encapsulating at least one a biological material and/or a bioactive molecule, the method comprising providing a composite biocompatible ceramic- based material of any one embodiments 1 to 10 or the ink of any one of embodiments 11 to 15, and at least one biological molecule and/or a bioactive molecule, and allowing the composite biocompatible ceramic-based material or the ink to set.

39. A method of promoting bone formation in a subject, the method comprising administering: a composite biocompatible ceramic-based material of any one embodiments 1 to 10, optionally comprising at least one bioactive molecule and/or biological molecule; or the ink of any one of embodiments 11 to 15, optionally comprising at least one bioactive molecule and/or at least one biological molecule, to the subject.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the above-described embodiments, without departing from the broad general scope of the present disclosure. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims

CLAIMS:

1. A composite biocompatible ceramic based material comprising particles of an inorganic salt of phosphoric acid, wherein the particles of the inorganic salt of phosphoric acid are dispersed in a mixture comprising an oil phase and a surfactant.

2. The composite biocompatible ceramic based material of claim 1, wherein the inorganic salt of phosphoric acid is alpha-tricalcium phosphate (a- TCP).

3. The composite biocompatible ceramic based material of claim 2 wherein the a- TCP is synthesised by mixing calcium carbonate and calcium hydrogen phosphate, optionally in a 1 :2 molar ratio.

4. The composite biocompatible ceramic based material of any one of claims 1 to 3, wherein the oil phase is glycerol.

5. The composite biocompatible ceramic based material of any one of claims 1 to 4 wherein the surfactant is a non-ionic surfactant.

6. The composite biocompatible ceramic based material of claim 5 wherein the non- ionic surfactant is polyoxyethylenesorbitan monooleate.

7. The composite biocompatible ceramic based material of any one of the preceding claims further comprising an accelerator.

8. The composite biocompatible ceramic based material of claim 7, wherein the accelerator is ammonium phosphate dibasic (APD).

9. The composite biocompatible ceramic based material of claim 7, wherein the accelerator is sodium phosphate dibasic.

10. The composite biocompatible ceramic based material of any one of the claims 1 to 9, wherein between about 50 and 70 wt.% of particles of inorganic salt of phosphoric acid is dispersed in a mixture of about 20 35 wt.% of the oil phase, about 5-10 wt.% of the surfactant and about 3-8 wt.% of an accelerator.

11 An ink for additive manufacturing, the ink composed of a composite biocompatible ceramic-based material which when deposited in or on a yield-stress support matrix in the presence of at least one bioactive molecule and/or biological material, and allows free-form three dimensional printing of ceramic bone mimetic shapes integrated with the at least one bioactive molecule and / biological material.

12. The ink of claim 11, wherein the composite biocompatible ceramic-based material comprises particles of an inorganic salt of phosphoric acid; the particles of inorganic salt of phosphoric acid being dispersed in a mixture comprising an oil phase and a surfactant.

13. The ink of claim 11 or claim 12, wherein the ink composed of a material which when deposited in or on a yield stress support matrix forms free-form three- dimensional printing of simple and complex ceramic bone mimetic shapes integrated with the at least one bioactive molecule and/or biological material.

14. The ink of any one of claims 11 to 13, wherein the yield stress support matrix is a suspension of gelatine microspheres.

15. The ink of any one of claims 11 to 13, wherein the composite biocompatible ceramic based material is the composite biocompatible ceramic based material of any one of claims 1 to 10.

16. A method of making a composite biocompatible ceramic based material, the method comprising mixing an oil phase, a surfactant and an accelerator together to form a suspension, adding particles of an inorganic salt of phosphoric acid to the suspension, and homogenising the mixture to form a paste of the ceramic based material.

17. A method of fabrication of a composite biocompatible ceramic material structure using an additive manufacturing process, the method comprising: depositing a composite biocompatible ceramic-based material in a yield- stress support matrix bath comprising at least one bioactive molecule and/or biological material, performing omnidirectional 3 dimensional printing of a structure composed of the biocompatible ceramic material integrated with the at least one bioactive molecule and/or biological material, wherein the yield-stress support matrix holds the printed ceramic material in place to obtain free-form printing of the structures.

18. The method of fabrication of a composite biocompatible ceramic material structure of claim 17, wherein the composite biocompatible ceramic-based material comprises particles of an inorganic salt of phosphoric acid, the particles of inorganic salt of phosphoric acid being dispersed in a mixture of an oil phase and a surfactant

19. The method of fabrication of a composite biocompatible ceramic material structure of claim 17 or claim 18, wherein the yield-stress support matrix is a suspension of gelatin microspheres.

20. The method of fabrication of a composite biocompatible ceramic material structure of claim 19, wherein the gelatin microspheres dissolve at about 37 °C.

21. The method of fabrication of a composite biocompatible ceramic material structure of any one of claims 16 to 20, wherein the at least one biological material is live cells.

22. The method of fabrication of a composite biocompatible ceramic material structure of any one of claims 16 to 21, wherein an anti-inflammatory medication is added to the biocompatible ceramic material before the step of printing.

23. The method of fabrication of a composite biocompatible ceramic material structure of any one of claims 16 to 22, wherein at least one bioactive molecule is added to the biocompatible ceramic material before the step of printing.

24. The method of fabrication of a composite biocompatible ceramic material structure of claim 23, wherein at least one bioactive molecule is fluorescein isothiocyanate labelled bovine serum albumin (FITC-BSA).

25. The method of fabrication of a composite biocompatible ceramic material structure of any one of claims 16 to 24, wherein fabrication is performed at about room temperature.

26. The method of fabrication of a composite biocompatible ceramic material, wherein the composite biocompatible ceramic material is defined by any one of claims 1 to 10

27. A bone like structure mimicking bone and formed by the method of any one of claims 16 to 26 using the composite biocompatible ceramic-based material of any one claims 1 to 10 or the ink of any one of claims 11 to 15.

28. The bone like structure of claim 27, wherein the structure is a high resolution construct with a hierarchical microstructure.

29. The bone like structure of claim 28, wherein the high-resolution construct is a multiphasic construct with hierarchical microstructure.

30. The bone like structure of claim 29, wherein the construct with hierarchical microstructure is an irregular bone structure mimicking one of a human osseous labyrinth, a trabecular bone or a helical haversian canal.

31. The bone like structure of any one of claims 27 to 30, wherein the structure is integrated with biological cells.

32. The bone like structure of any one of claims 27 to 31, wherein the structure incorporates at least one bioactive molecule and/or biological molecule.

33. The bone like structure of any one of claims 27 to 32, further comprising one or more drugs supporting sustained drug release.

34. The bone like structure of claim 33, wherein the drugs are selected from dexamethasone and ibuprofen.

35. The bone like structure of claim 32, wherein the bone like structure comprises a protein or fluorescein isothiocyanate labelled bovine serum albumin (FITC-BSA).

36. A method of using the composite biocompatible ceramic-based material of any one claims 1 to 10 or the ink of any one of claims 11 to 15 as a bone cement.

37. The method of claim 36, wherein the bone cement is used to bond two or pieces of bone together and/or bond one or more synthetic materials to bone.

38. A method of encapsulating at least one a biological material and/or a bioactive molecule, the method comprising providing a composite biocompatible ceramic- based material of any one claims 1 to 10 or the ink of any one of claims 11 to 15, and at least one biological molecule and/or a bioactive molecule, and allowing the composite biocompatible ceramic-based material or the ink to set.

39. A method of promoting bone formation in a subject, the method comprising administering: a composite biocompatible ceramic-based material of any one claims 1 to 10, optionally comprising at least one bioactive molecule and/or biological molecule; or the ink of any one of claims 11 to 15, optionally comprising at least one bioactive molecule and/or at least one biological molecule, to the subject.