WO2025058569A2

WO2025058569A2 - Calcium phosphate scaffold and methods thereof

Info

Publication number: WO2025058569A2
Application number: PCT/SG2024/050586
Authority: WO
Inventors: Yujun Tan; Pengyu Chen; Huiyu Wang
Original assignee: National University of Singapore
Current assignee: National University of Singapore
Priority date: 2023-09-15
Filing date: 2024-09-13
Publication date: 2025-03-20
Anticipated expiration: 2026-03-15
Also published as: WO2025058569A3; WO2025058569A9

Abstract

The present invention discloses a method of fabricating biphasic calcium phosphate 5 product, comprising reacting calcium carbonate with phosphoric acid to obtain a calcium phosphate powder; the calcium phosphate powder with at least one binder and optionally an additional calcium source to obtain a bio-ink; 3D printing a calcium phosphate structure using the bio-ink; and sintering the calcium phosphate structure to obtain the biphasic calcium phosphate product; wherein the biphasic calcium phosphate 10 material comprises hydroxyapatite (HA) and beta-tricalcium phosphate (β-TCP); wherein a final mole ratio of HA to β-TCP is dependent on a final calcium/phosphorus (Ca/P) ratio of the calcium phosphate powder or the bio-ink. The present invention also discloses a method of fabricating a calcium phosphate product, a biphasic calcium phosphate product, a calcium phosphate powder and a bio-ink.

Description

Calcium Phosphate Scaffold and Methods Thereof

Technical Field

The present invention relates, in general terms, to a biphasic calcium phosphate product and a method of fabricating a biphasic calcium phosphate product.

Background

Autologous bone harvested from the patient's own bone is the gold standard bone substitute for repairing large bone defects. However, the amount of autologous bone harvestable from a patient is limited and the bone subtraction itself poses significant health risks and results in loss of structural integrity of the remaining bone.

Bone tissue engineering has drawn extensive attention for repairing critical-size bone defects, offering a promising alternative to conventional methods such as bone grafting using autografts or allografts. Developments in tissue engineering have provided synthetic implants, for instance in the form of scaffold materials, which allow attachment of bone cells and ingrowth of new bone tissue and subsequent deposition of new bone mineral. The synthetic materials may either be grafted ex vivo with bone cells prior to implantation or may be implanted as naked scaffolds that attract bone cells from the periphery to the site of the implant.

The material should be physiologically acceptable, so as to avoid the initiation of clots, inflammatory response, and the like. In addition, the material must be strong and not friable. Furthermore, there should be strong adhesion between the material and any remaining bone. Besides the biological and physiological considerations, there are the additional considerations of how the material is made and the ease with which it may be formed to a desired shape.

Recent advances in tissue engineering have produced a variety of valuable scaffold materials. Calcium phosphates such as hydroxyapatite (HA; the mineral phase of bone), biphasic calcium phosphate (BCR) and alpha- or beta-tricalcium phosphate (TCP) are known to possess both osteoconductive (bioactive) as well as osteoinductive properties and provide very suitable scaffold materials. The bioactive nature of calcium phosphates allows them to function as a template for new bone formation by osteogenic cells through deposition of new mineral material at the scaffold's surface and is an important feature of the scaffold material. The osteoinductive nature of calcium phosphates is a qualitative feature, i.e. the capacity to induce the development of the new bone tissue, thereby enhancing the rate of deposition of new mineral depends on various material parameters. Bone induction is generally defined as the mechanism by which a mesenchymal tissue is induced to change its cellular structure to become osteogenic. In general, porous calcium phosphates have been found to exhibit osteoi nd activity.

In bone tissue engineering, a porous 3D scaffold mimicking the structure of natural extracellular matrix (ECM) is used as a template for cell growth and tissue regeneration. Currently, HA and (β-TCP are the two most investigated materials to build the bone scaffold due to their excellent biocompatibility, excellent osteointegration, osteoconductive and osteoinductive properties. Although there are extensive studies on the synthesis, fabrication, biological responses, and biodegradation behaviour of HA/|3- TCP scaffolds, complex synthesis process and costly reagents were used in the process.

It would be desirable to overcome or ameliorate at least one of the above-described problems.

Summary

The present disclosure concerns a method of fabricating a biphasic calcium phosphate product, comprising : a) reacting calcium carbonate with phosphoric acid to obtain a calcium phosphate powder; b) combining the calcium phosphate powder with at least one binder and optionally an additional calcium source to obtain a bio-ink; c) 3D printing a calcium phosphate structure using the bio-ink; and d) sintering the calcium phosphate structure to obtain the biphasic calcium phosphate product; wherein the biphasic calcium phosphate product comprises hydroxyapatite (HA) and beta-tricalcium phosphate ((β-TCP); and wherein a final mole ratio of HA to (β-TCP is dependent on a final calcium/phosphorus (Ca/P) ratio of the calcium phosphate powder or the bio-ink. In some embodiments, the final mole ratio of calcium to phosphorus (after step b)) is about 1.3 and about 1.9. In some embodiments, the final mole ratio is about 1.5 and about 1.7.

In some embodiments, the final mole ratio of HA to (β-TCP is about 1 : 100 to about 100: 1.

The present disclosure concerns a method of fabricating a calcium phosphate product, comprising : a) reacting calcium carbonate with phosphoric acid to obtain a calcium phosphate powder; b) combining the calcium phosphate powder with at least one binder and optionally an additional calcium source to obtain a bio-ink; c) 3D printing a calcium phosphate structure using the bio-ink; and d) sintering the calcium phosphate structure to obtain the calcium phosphate product; wherein the calcium phosphate product comprises beta-tricalcium phosphate ([β-TCP); wherein a final mole ratio of calcium to phosphorus is about 1.3 to about 1.6.

In some embodiments, the final mole ratio of calcium to phosphorus is about 1.4 to about 1.5.

The present disclosure concerns a method of fabricating a calcium phosphate product, comprising : a) reacting calcium carbonate with phosphoric acid to obtain a calcium phosphate powder; b) combining the calcium phosphate powder with at least one binder and optionally an additional calcium source to obtain a bio-ink; c) 3D printing a calcium phosphate structure using the bio-ink; and d) sintering the calcium phosphate structure to obtain the calcium phosphate product; wherein the calcium phosphate product comprises hydroxyapatite (HA); wherein a final mole ratio of calcium to phosphorus is about 1.6 to about 1.9. In some embodiments, the final mole ratio of calcium to phosphorus is about 1.7 to about 1.9.

In some embodiments, the calcium carbonate is obtained from a naturally occurring source. In some embodiments, the naturally occurring source is selected from coral bones, animal bones, bivalves, eggshells and gastropod shells.

In some embodiments, the method further comprises a step before step a) of ball milling the calcium carbonate, the ball milled calcium carbonate characterised by a particle size distribution with about 30% to about 50% of particles having a particle size of less than about 1 μm. In some embodiments, the ball milled calcium carbonate is characterised by a particle size distribution with about 45% of particles having a particle size of less than about 1 μm.

In some embodiments, the ball milled calcium carbonate is characterised by a particle size of about 0.5 μm to about 10 μm.

In some embodiments, the ball milling is characterised by a speed of about 200 rpm to about 600 rpm for about 30 minutes to about 4 hours. In some embodiments, the ball milling is characterised by a speed of about 400 rpm for about 2 hours.

In some embodiments, the phosphoric acid is selected from phosphorus oxoacids. In some embodiments, the phosphorus oxoacids is selected from orthophosphoric acid (H3PO4), pyrophosphoric acid (H4P2O7), hypophosphoric acid (H4P2O6), hypophosphorus acid (H3PO2), phosphorus acid (H3PO3), triphosphoric acid (H5P3O10), peroxomonophosphoric acid (H3PO5), or peroxodiphosphoric acid (H4P2O8). In some embodiments, the phosphoric acid is orthophosphoric acid.

In some embodiments, the phosphoric acid is a phosphoric acid solution characterised by a wt% of about 10 wt% to about 20 wt% of phosphoric acid relative to the phosphoric acid solution. In some embodiments, the wt% is about 16 wt%.

In some embodiments, step a) further comprises stirring the calcium carbonate with phosphoric acid at a temperature of about 50 °C to about 100 °C and at a stirring speed of about 200 rpm to about 1000 rpm for about 0.5 hour to about 30 hours. In some embodiments, the stirring is performed at a temperature of about 80 °C and at a stirring speed of about 400 rpm to about 800 rpm for about 24 hours.

In some embodiments, the stirring is mechanical stirring.

In some embodiments, step a) further comprises drying of the calcium phosphate powder at a temperature of about 50 °C to about 100 °C for about 8 hours to about 24 hours. In some embodiments, step a) further comprises drying of the calcium phosphate powder at a temperature of about 70 °C for about 12 hours.

In some embodiments, the method further comprises a step after a) of ball milling the calcium phosphate powder. In some embodiments, the ball milled calcium phosphate powder characterised by a particle size distribution with about 35% to about 60% of particles having a particle size of less than about 1 μm. In some embodiments, the ball milled calcium phosphate powder is characterised by a particle size distribution with about 54% of particles having a particle size of less than about 1 μm.

In some embodiments, the ball milled calcium phosphate powder is characterised by a particle size distribution of about 0.5 μm to about 5 μm.

In some embodiments, the ball milling is characterised by a speed of about 200 rμm to about 600 rμm for about 30 minutes to about 4 hours. In some embodiments, the ball milling is characterised by a speed of about 400 rpm for about 1 hour.

In some embodiments, the method further comprises adding additional calcium source to the calcium phosphate powder after step a).

In some embodiments, the additional calcium source is calcium carbonate.

In some embodiments, the biphasic calcium phosphate product and/or calcium phosphate product is characterised by a calcium phosphate to calcium oxide ratio of about 1.1: 1 to about 2.5:1.

In some embodiments, the bio-ink is characterised by a wt% of about 30 wt% to 60 wt% of calcium phosphate powder relative to a total composition of the bio-ink. In some embodiments, the bio-ink is characterised by a wt% of about 40 wt% to about 50 wt% of calcium phosphate powder relative to a total composition of the bio-ink.

In some embodiments, the bio-ink further comprises a solvent. In some embodiments, the solvent is selected from ethyl acetate, acetic acid, ethanol and water. In some embodiments, the solvent is water.

In some embodiments, the binder is selected from polyvinyl alcohol (PVA), polylactic acid (PLA), poly lactic-co-glycolic acid (PLGA), polycaprolactone (PCL) and a combination thereof. In some embodiments, the binder is polyvinyl alcohol.

In some embodiments, the bio-ink is characterised by a storage modulus greater than a loss modulus at a shear strain of less than about 0.5%, and a loss modulus greater than a storage modulus at a shear strain of more than 0.5%.

In some embodiments, the bio-ink exhibits shear-thinning behaviour.

In some embodiments, the 3D printing is direct ink writing.

In some embodiments, the method is characterised by a printing pressure of about 40 kPa to about 400 kPa. In some embodiments, the printing pressure is about 50 kPa to about 300 kPa.

In some embodiments, the method is characterised by a printing speed of about 1 mm/s to about 20 mm/s. In some embodiments, the printing speed is about 5 mm/s to about 15 mm/s.

In some embodiments, the sintering step is performed at a heating rate of about 1 °C/min to about 10 °C/min to a temperature of about 600 °C to about 1000 °C and maintained for a duration of about 0.5 hour to about 4 hours, and subsequently at a temperature of about 1000 °C to about 1800 °C for a duration of about 0.5 hour to about 10 hours.

In some embodiments, the biphasic calcium phosphate product comprises HA particles and β-TCP particles sintered to each other, the particles characterised by an average particle size of about 0.5 μm to about 5 μm, preferably about 1 μm to about 3 μm.

In some embodiments, the biphasic calcium phosphate product and/or calcium phosphate product comprises macropores and/or micropores, the macropores having a macropore size of about 200 μm to about 1500 μm and the micropores having a micropore size of about 0.5 μm to about 15 μm. In some embodiments, the macropores have a macropore size of about 300 μm to about 500 μm and the micropores have a micropore size of about 1 μm to about 10 μm.

In some embodiments, the biphasic calcium phosphate product and/or calcium phosphate product is characterised by a porosity of about 30% to about 95%. In some embodiments, the porosity is about 50% to about 90%.

In some embodiments, the biphasic calcium product and/or calcium phosphate product is characterised by a compressive strength of about 2 MPa to about 15 MPa. In some embodiments, the compressive strength is about 3 MPa to about 11 MPa.

In some embodiments, the biphasic calcium product and/or calcium phosphate product is characterised by a Young's modulus of about 45 MPa to about 150 MPa. In some embodiments, the Young's modulus is about 51 MPa to about 125 MPa.

In some embodiments, the biphasic calcium product and/or calcium phosphate product is characterised by a cell viability of about 90% to about 100%.

The present disclosure also concerns a biphasic calcium phosphate product and/or calcium phosphate product fabricated according to the method as disclosed herein.

The present disclosure also concerns a biphasic calcium phosphate product comprising: a) hydroxyapatite (HA) particles and beta-tricalcium phosphate (β-TCP) particles sintered to each other, the particles having an average particle size of about 0.5 μm to about 5 μm; wherein the biphasic calcium phosphate product is characterised by micropores having a pore size of about 0.5 μm to about 15 μm; wherein the biphasic calcium phosphate product is characterised by macropores having a pore size of about 200 μm to about 1500 μm; and wherein the biphasic calcium phosphate product is characterised by a porosity of about 30% to about 95%.

The present disclosure also concerns a calcium phosphate product comprising: beta-tricalcium phosphate (β-TCP) particles, the particles having an average particle size of about 0.5 μm to about 5 μm; wherein the calcium phosphate product is characterised by micropores having a pore size of about 0.5 μm to about 15 μm; wherein the calcium phosphate product is characterised by macropores having a pore size of about 200 to about 1500 μm; wherein the calcium phosphate product is characterised by a porosity of about 30% to about 95%.

The present disclosure also concerns a calcium phosphate product comprising: hydroxyapatite (HA) particles, the particles having an average particle size of about 0.5 μm to about 5 μm; wherein the calcium phosphate product is characterised by micropores having a pore size of about 0.5 μm to about 15 μm; wherein the calcium phosphate product is characterised by macropores having a pore size of about 200 to about 1500 μm; wherein the calcium phosphate product is characterised by a porosity of about 30% to about 95%.

The present disclosure also concerns a calcium phosphate powder; wherein the calcium phosphate powder is characterised by a calcium/phosphorus ratio is about 0.5 to about 1.9; wherein the calcium phosphate powder is characterised by a particle size of about 0.5 μm to about 5 μm; and wherein the calcium is obtained from a naturally occurring source.

The present disclosure also concerns a bio-ink, comprising: a) calcium phosphate powder; and b) a binder; wherein the calcium phosphate powder is characterised by a calcium/phosphorus ratio is about 1.3 to about 1.9; and wherein the bio-ink is characterised by a wt% of about 30 wt% to 60 wt% of calcium phosphate powder relative to a total composition of the bio-ink.

In some embodiments, the calcium phosphate powder is disclosed herein.

The present invention also discloses a method of implanting a calcium scaffold in a subject in need thereof, wherein the calcium scaffold comprises a biphasic calcium phosphate product and/or a calcium phosphate product as disclosed herein.

In some embodiments, the biphasic calcium phosphate product and/or calcium phosphate product is biodegradable in order to allow regeneration of bone tissue.

Brief description of the drawings

Embodiments of the present invention will now be described, by way of non-limiting example, with reference to the drawings in which:

Figure 1 shows the fabrication process of nature-derived 3D-printed biphasic scaffold for bone tissue engineering.

Figure 2 shows the particle size distribution of (a) ball-milled coral powder and (b) - printed scaffolds (c) before sintering and (d) after sintering.

Figure 3 shows XRD patterns of (a) ball-milled coral powder, (b) coral powder after reaction with phosphoric acid at a Ca/P ratio at 0.5, (c) sintered scaffold with a Ca/P ratio of 1.6, and (d) sintered scaffold with a Ca/P ratio of 1.7.

Figure 4 shows XRD patterns of further examples, in which (a) ball-milled coral powder, (b) sintered scaffold with a Ca/P ratio of 1.3, (c) sintered scaffold with a Ca/P ratio of 1.56, and (d) sintered scaffold with a Ca/P ratio of 1.7.

Figure 5 shows optical images of the 3D-printed scaffolds (a) before sintering and (b) after sintering. SEM images of the 3D-printed scaffolds (c) before sintering and (d) after sintering.

Figure 6 shows 3D models of the designed microlattice, optical images of the macropores in the 3D-printed scaffolds after sintering and SEM images of the filament surface in the 3D-printed scaffold A, B, and C after sintering. Scaffold A, B and C are sintered scaffolds with a Ca/P ratio of 1.5.

Figure 7 shows optical and SEM images obtained from another example of the sintered HA scaffold and sintered |β-TCP scaffold, (a) Optical image of the sintered HA scaffold, (b) SEM images of the surface of the HA scaffold, (c) SEM images of the cross-section of the HA scaffold, (d) Optical image of the sintered β-TCP scaffold, (e) SEM images of the surface of the (β-TCP scaffold, (f) SEM images of the cross-section of the (β-TCP scaffold.

Figure 8 shows stress-strain curves, compressive stress and Young's modulus of the 3D-printed bone scaffolds, (a) Stress-strain curves, (b) compressive stress, and (c) Young's modulus of the 3D-printed bone scaffolds.

Figure 9 shows stress-strain curves, compressive stress and Young's modulus of further examples of the 3D-printed bone scaffolds, (a) Stress-strain curves, (b) compressive stress, and (c) Young's modulus of the 3D-printed bone scaffolds.

Figure 10 shows LIVE/DEAD staining results and CCK-8 assay results on the 3D-printed scaffolds, (a-c) LIVE/DEAD staining of MC3T3-E1 cultured on the 3D-printed scaffolds for 1, 2, and 3 days, (d) CCK-8 assay of MC3T3-E1 cultured on the 3D-printed scaffolds for 1, 2, and 3 days.

Figure 11 shows LIVE/DEAD staining results and CCK-8 assay results on the 3D-printed scaffolds, (a) CCK-8 assay result of MC3T3-E1 preosteoblasts cultured on HA and [β-TCP scaffolds at day2, day4 and day6. (b) LIVE/DEAD staining of MC3T3-E1 preosteoblasts after 14 days of culture on HA and [β-TCP scaffolds.

Figure 12 shows the viscosity profile of a hydroxyapatite (HA) bio-ink. (a) Viscosity profile as a function of shear rate for ceramic bio-ink with a 46 wt% solid loading, (b) Log - log plot of the storage modulus G' and loss modulus G" as a function of shear strain for the same ink.

Figure 13 shows micro-CT scan results of 1 month implantation in rabbits, (a) Femoral condyle sample implanted with 3D printed HA scaffold for one month, (b) Femoral condyle sample implanted with 3D printed [β-TCP scaffold for one month, (c) Reconstructed micro-CT images of femoral condyle sample implanted with 3D printed HA scaffold for one month, (d) Reconstructed micro-CT images of femoral condyle sample implanted with 3D printed [β-TCP scaffold for one month.

Detailed description

The present disclosure is predicated on the understanding that HA and (β-TCP can be obtained starting from natural CaCOs, which is the main constituent of coral bones, animal bones and shells. These natural sources of CaCC₃ are usually considered as waste products with little economic value. Using natural CaCOs sources can significantly cut down the cost of fabricating bone scaffolds. In addition, the hierarchical porous structures of those natural materials can enhance the bioactivities of the nature-derived scaffolds.

Earlier work has shown that nature-derived HA powder may be synthesised by reacting utilized cuttlebones, eggshells, and mussel shells with H3PO4 and fabricate a composite scaffold with polycaprolactone (PCL) using 3D printing technique. This nature-derived PCL/HA scaffold demonstrated a high elastic modulus of 117 -317 MPa, which are comparable with trabecular bones. In addition, the composite PCL/HA scaffold showed improved bioactivity compared to pure PCL scaffolds. Another work reacts eggshell powders with HNO3 and (NH^ZHPOA to produce HA powders, which were subsequently mixed with PCL and dichloromethane to produce to a viscous bio-ink for 3D printing. The printed scaffold were able to support cell attachment and spreading.

While the studies mentioned above effectively converted natural CaCOa sources into bioactive HA scaffolds and showcased their remarkable bioactivity, the potential applications of these scaffolds remain constrained due to their exclusive composition of the HA phase. HA is mechanically stiff and presents a slow biodegradation rate, thus limiting its versatility. β-TCP, on the other hand, possess a lower mechanical stiffness and faster biodegradation and bioabsorption compared to HA. Hence, a scaffold with both HA and β-TCP may provide a better bioabsorption while still maintaining the required mechanical stiffness for a bone scaffold.

The present disclosure concerns a method of fabricating a biphasic calcium phosphate product. The biphasic calcium phosphate product comprises hydroxyapatite (HA) and beta-tricalcium phosphate (β-TCP). The biphasic calcium phosphate product may be biocompatible and osteoconductive for bone tissue repair. The biphasic calcium phosphate product may be implantable into a patient for bone repair. The ratio of HA and β-TCP may be altered depending on the specific bone types and applications. HA is known to be stronger but degrades slower, while β-TCP may be weaker but with faster resorption. The ratio of HA and β-TCP may be controlled by adjusting the calcium/phosphorus mole ratio in the reaction system, allowing for tunable biodegradation rate and mechanical stiffness for various bone types and age groups to meet the different strength and regeneration requirements. This allows for a personalised solution for different patients who require different types of bone, which may be achievable via the different HA/ β-TCP ratio.

Calcium phosphate refers to a family of materials and minerals containing calcium ions and phosphorus anions. Calcium phosphates include various salts of tribasic phosphoric acid (H₃PO₄). H₂PO₄, HPCU²', PO4³' ions can all be formed through progressive removal of H⁺ ions from this acid. Calcium phosphate are found in many living organisms, such as bone mineral and tooth enamel. They are known as the major inorganic material in approximately 60% of all native human bones. The release of calcium and phosphorus ions regulates the activation of osteoblasts and osteoclasts to facilitate bone regeneration. Calcium is one of the ions that form the bone matrix, and it exists mostly in the form of calcium phosphates in bone tissues. These calcium ions cause bone formation and maturation through calcification. In addition, calcium ions affect bone regeneration through cellular signaling. Calcium stimulates mature bone cells through the formation of nitric oxide and induces bone growth precursor cells for bone tissue regeneration. Phosphorus ions are present in the human body in large amounts. They are involved in a variety of substances such as proteins, nucleic acid, and adenosine triphosphate, and they affect physiological processes. Over 80% of phosphorous ions are present in bone in the form of calcium phosphates along with calcium ions. Phosphorous mainly exists in the form of phosphate (PO4^3-), which has great influence on tissue formation and growth.

Calcium phosphate-based biomaterials are stable, biocompatible, and osteoconductive and thus increase the scope of tissue generation. The control of surface properties and porosity of calcium phosphate affects cell and protein adhesion and growth and regulates bone mineral formation. Properties affecting bioactivity vary depending on the types of calcium phosphates such as HA, TCP and may be utilized in various applications because of differences in ion release, solubility, stability, and mechanical strength.

HA is a naturally occurring mineral form of calcium apatite and is characterised by the chemical formula Ca₅(PO₄) ₃(OH), although it is often written Ca₁₀(PO₄)₆(OH)₂ to denote that the crystal unit cell comprises two molecules. Hydroxyapatite with a Ca/P mole ratio of 1.67 is the main structural component of tooth enamel and bone mineral that provides hardness. HA is the most stable calcium phosphate with low solubility in physiological environments defined by temperature, pH, body fluids, etc. and the surface of HA may act as a nucleating site for bone minerals in body fluids. In addition, HA does not cause inflammatory reactions when applied clinically.

TCP is a tertiary calcium phosphate with the chemical formula Ca₃(PO₄)₂. It exists as three crystalline polymorphs a, a', and β. The a and a' states are stable at high temperatures. a-TCP and a'-TCP can be formed at 1125 °C or higher, and [β-TCP is formed at a temperature of 900-1100 °C. (β-TCP has a more stable structure and higher biodegradation rate than those of a-TCP. Therefore, |β-TCP is generally used in bone regeneration. [β-TCP is less stable than HA but has a faster degradation rate and higher solubility. In addition, it has a high resorption rate and is widely used to increase biocompatibility. (β-TCP promotes the proliferation of osteoprecursor cells such as osteoblasts and bone marrow stromal cells. These properties are due to the excellent biomineralization and cell adhesion by the nanoporous structure of [β-TCP.

As used herein, biphasic calcium phosphates (BCP) are composed of a combination of HA and [β-TCP and may be used in bone regeneration procedures. BCP may have different ratios of HA/ [β-TCP, giving rise to balanced phases of activity, a more stable phase of HA, and a more soluble phase of [β-TCP.

Without wanting to be bound by theory, it was found that the solubility of calcium phosphate phases in aqueous solution correlated with the Ca/P mole ratio. Generally the higher the Ca/P mole ratio, the lower is the solubility. The resorption rate is dependent on the HA/ [ β-TCP ratio, where a higher [β-TCP amount means a higher solubility. The higher ratio of HA to β-TC P may be suitable for bone regeneration in dehiscence types of defects and surgically created periodontal defects. BCP may gradually dissolves in the body, determining the new bone formation by the release of calcium and phosphate ions. BCPs are highly biocompatible, and they do not provoke a foreign body or a toxic response. The mixture of HA and [ β-TCP may stimulate the osteogenic differentiation of mesenchymal stem cells, increase cell adhesion, attach growth factors, and enhance mechanical properties.

Accordingly, the present disclosure concerns a method of fabricating a biphasic calcium phosphate product, comprising: a) reacting calcium carbonate with phosphoric acid to obtain a calcium phosphate powder; b) combining the calcium phosphate powder with at least one binder and optionally an additional calcium source to obtain a bio-ink; c) 3D printing a calcium phosphate structure using the bio-ink; and d) sintering the calcium phosphate structure to obtain the biphasic calcium phosphate product; wherein the biphasic calcium phosphate product comprises hydroxyapatite (HA) and beta-tricalcium phosphate ( β-TCP); wherein a mole ratio of HA to [β-TCP is dependent on a final calcium/phosphorus (Ca/P) ratio of the calcium phosphate powder or the bio-ink.

The method is illustrated in Figure 1.

Calcium carbonate reacts with phosphoric acid to produce calcium phosphate, carbon dioxide and water. The phosphoric acid source may be 16 wt% phosphoric acid solution. The calcium carbonate powder and phosphoric acid may be mixed together such that the final Ca/P mole ratio is about 1.3 to about 1.9. The calcium phosphate powder formed may comprise particles comprising different wt% of phosphorus, which upon sintering of the powder results in a biphasic calcium phosphate material. Single phase HA material or single phase β-TCP material may also be formed at the higher and lower ratios respectively.

In some embodiments, a mole ratio of calcium to phosphorus in step a) is about 1.3 and about 1.9. In other embodiments, the mole ratio of calcium to phosphorus is about 1.3 to about 1.8, about 1.3 to about 1.7, about 1.3 to about 1.6, about 1.3 to about 1.5, about 1.4 to about 1.8, about 1.4 to about 1.7, about 1.4 to about 1.6 about 1.4 to about 1.5, about 1.5 to about 1.9, about 1.5 to about 1.8, about 1.5 to about 1.7, about 1.5 to about 1.6, about 1.6 to about 1.9, about 1.6 to about 1.8, or about 1.6 to about 1.7. In some embodiments, the mole ratio of calcium to phosphorus in step a) is about 1.5 and about 1.7.

In some embodiments, a final mole ratio of calcium to phosphorus (after step b)) is about 1.3 to about 1.9. In this regard, the calcium includes the calcium carbonate of step a) and the additional calcium source. In other embodiments, the final mole ratio is about 1.3 to about 1.8, about 1.3 to about 1.7, about 1.3 to about 1.6, about 1.3 to about 1.5, about 1.4 to about 1.8, about 1.4 to about 1.7, about 1.4 to about 1.6 about 1.4 to about 1.5, about 1.5 to about 1.9, about 1.5 to about 1.8, about 1.5 to about 1.7, about 1.5 to about 1.6, about 1.6 to about 1.9, about 1.6 to about 1.8, or about 1.6 to about 1.7. In some embodiments, the final mole ratio is about 1.5 to about 1.7. In some embodiments, the final mole ratio is about 1.67.

In some embodiments, the final mole ratio of HA to (β-TCP is about 1 : 100 to about 100: 1. In other embodiments, the final mole ratio of HA to (β-TCP is about 1:100 to about 2: 1, about 1 :100 to about 1 :1, about 1: 100 to about 1:5, about 1: 100 to about 1 : 10, about 1: 100 to about 1 :50, about 1:100 to about 1:75, about 1 :75 to about 100: 1, about 1:75 to about 2: 1, about 1 :75 to about 1:1, about 1:75 to about 1:5, about 1 :75 to about 1: 10, about 1:75 to about 1 :50, about 1:50 to about 100: 1 about 1 :50 to about 2:1, about 1:50 to about 1 : 1, about 1:50 to about 1 :5, about 1:50 to about 1: 10, about 1 : 10 to about 100:1, about 1: 10 to about 2: 1, about 1 :10 to about 1 : 1, about 1 : 10 to about 1:5, about 1:5 to about 100:1, about 1:5 to about 2: 1, about 1 :5 to about 1:1, about 1:1 to about 100: 1, or about 1:1 to about 2:1. In some embodiments, the final mole ratio of HA to (β-TCP is about 1 :100 to about 1 : 1. In some embodiments, the final mole ratio of HA to (β-TCP is about 1 : 1 to about 100:1.

The present disclosure also concerns a method of fabricating a calcium phosphate product, comprising : a) reacting calcium carbonate with phosphoric acid to obtain a calcium phosphate powder; b) combining the calcium phosphate powder with at least one binder and optionally an additional calcium source to obtain a bio-ink; c) 3D printing a calcium phosphate structure using the bio-ink; and d) sintering the calcium phosphate structure to obtain the calcium phosphate product; wherein the calcium phosphate product comprises beta-tricalcium phosphate ([β-TCP); and wherein a final mole ratio of calcium to phosphorus is about 1.3 to about 1.6.

In some embodiments, calcium phosphate product consist essentially of beta-tricalcium phosphate ((β-TCP). In some embodiments, calcium phosphate product consist of beta- tricalcium phosphate ((β-TCP). (β-TCP has a faster resorption compared to HA and may be more suited for a particular bone type and application. In some embodiments, a mole ratio of calcium to phosphorus in step a) is about 1.3 to about 1.6. In other embodiments, the mole ratio is about 1.3 to about 1.55, about 1.3 to about 1.5, about 1.35 to about 1.6, about 1.35 to about 1.55, about 1.35 to about

1.5, about 1.4 to about 1.6, about 1.4 to about 1.55, about 1.4 to about 1.5, about 1.45 to about 1.6, about 1.45 to about 1.55, about 1.45 to about 1.5, about 1.5 to about

1.6, or about 1.5 to about 1.55. In some embodiments, the mole ratio is about 1.4 to about 1.5, or about 1.45 to about 1.5. In some embodiments, the mole ratio is less than about 1.5.

In some embodiments, the final mole ratio of calcium to phosphorus (Ca/P) (after step b)) for forming a single phase β-TCP material is about 1.3 to about 1.6. In other embodiments, the final mole ratio is about 1.3 to about 1.55, about 1.3 to about 1.5, about 1.35 to about 1.6, about 1.35 to about 1.55, about 1.35 to about 1.5, about 1.4 to about 1.6, about 1.4 to about 1.55, about 1.4 to about 1.5, about 1.45 to about 1.6, about 1.45 to about 1.55, about 1.45 to about 1.5, about 1.5 to about 1.6, or about 1.5 to about 1.55. In some embodiments, the final mole ratio is about 1.4 to about 1.5, or about 1.45 to about 1.5. In some embodiments, the final mole ratio is less than about 1.5.

The present disclosure concerns a method of fabricating a calcium phosphate product, comprising : a) reacting calcium carbonate with phosphoric acid to obtain a calcium phosphate powder; b) combining the calcium phosphate powder with at least one binder and optionally an additional calcium source to obtain a bio-ink; c) 3D printing a calcium phosphate structure using the bio-ink; and d) sintering the calcium phosphate structure to obtain the calcium phosphate product; wherein the calcium phosphate product comprises hydroxyapatite (HA); wherein a final mole ratio of calcium to phosphorus is about 1.6 to about 1.9.

In some embodiments, calcium phosphate product consist essentially hydroxyapatite (HA). In some embodiments, calcium phosphate product consist of hydroxyapatite (HA). HA has a higher bioactivity and osteoconductivity compared to β-TCP, and may better promote bone cell attachment, proliferation and new bone formation.

In some embodiments, a mole ratio of calcium to phosphorus in step a) is about 1.6 to about 1.9. In other embodiments, the mole ratio is about 1.6 to about 1.8, about 1.6 to about 1.7, about 1.7 to about 1.9, or about 1.7 to about 1.8. In some embodiments, the mole ratio is about 1.7 to about 1.8.

In some embodiments, the final mole ratio of calcium to phosphorus (Ca/P) (after step b)) for forming a single phase HA material is about 1.6 to about 1.9. In other embodiments, the final mole ratio is about 1.6 to about 1.8, about 1.6 to about 1.7, about 1.7 to about 1.9, or about 1.7 to about 1.8. In some embodiments, the final mole ratio is about 1.7 to about 1.8.

It was found that the mechanical properties of the HA formed at various ratios of calcium to phosphorus between about 1.7 to about 1.8 may differ due to impurities. For example, a final mole ratio of calcium to phosphorus of about 1.7 may yield a stronger HA product.

In some embodiments, the biphasic calcium phosphate structure and/or calcium phosphate structure is a scaffold. Scaffolds provide structural support for cell attachment and subsequent tissue development. The scaffold may provide stable properties and allow for control of porosity and biocompatibility. The pore size of the scaffold improves revascularisation and bone remodelling, enabling the ingrowth of cells and proteins, enhancing biocompatibility and making them suitable for implant use.

In some embodiments, the calcium carbonate is obtained from a naturally occurring source. The calcium carbonate may be in calcite or aragonite forms. The naturally occurring source may be an organism-based source such shells of organisms such as bivalves, eggshells and gastropod shells, animal bones and coral bones. Obtaining the calcium carbonate from a naturally occurring source enables recycling of natural waste and is a cost-effective way as compared to synthesising calcium carbonate. Natural calcium carbonate sources have natural microstructures that may create micropores in the sintered biphasic calcium phosphate material. The porosity of the biphasic calcium phosphate material has an effect on bioactivity. An increase in porosity improves contact with bodily fluids on the surface area of the material. Dissolution rate may be enhanced and the presence of pores on the surface affects protein adsorption. Naturally occurring calcium carbonate may be gastropod shells (e.g. snail shells), eggshells, animal bones, coral bones and bivalves (e.g. oyster shells, mussel shells, scallop shells, cockles shells, clam shells, and abalone shells). The natural calcium carbonate sources may comprise 95% calcium carbonate, and may be boiled and cleaned to remove any remaining organic matter. The calcium carbonate content may be characterised using XRD/EDS and a Ca/P ratio is calculated for the reaction with phosphoric acid based on the characterised calcium carbonate content. In some embodiments, the naturally occurring source is selected from coral bones, animal bones, bivalves, eggshells and gastropod shells.

Ball milling is a grinding method where the grinder is filled with grinding balls. Ball milling is used to grind or blend materials for use in mineral dressing processes, paints, ceramics, and sintering processes. Size reduction is done by impact as the balls drop onto the material. Ball mills are often used to reduce the particle size, eliminate agglomeration, change the shape of particles, provide for mechanical alloying, mixing, producing powders and changing materials properties. Accordingly, in some embodiments, the method further comprises a step before step a) of ball milling the calcium carbonate.

In some embodiments, the ball milled calcium carbonate is characterised by a particle size distribution with about 30% to about 50% of particles having a particle size of less than about 1 μm. In other embodiments, the calcium carbonate is characterised by about 30% to about 45%, about 30% to about 40%, about 30% to about 35%, about 35% to about 50%, about 35% to about 45%, about 35% to about 40%, about 40% to about 50%, or about 40% to about 45% of particles having a particle size of less than about 1 μm. In some embodiments, the calcium carbonate is characterised by about 45% of particles having a particle size of less than 1 μm.

In some embodiments, the method further comprises a step before step a) of ball milling the calcium carbonate, the ball milled calcium carbonate characterised by a particle size distribution with about 45% of particles having a particle size of less than about 1 μm.

In some embodiments, the ball milled calcium carbonate is characterised by a particle size of about 0.5 μm to about 10 μm. In other embodiments, the particle size is about 0.5 μm to about 8 μm, about 0.5 μm to about 5 μm, about 0.5 μm to about 2 μm, about 1 μm to about 10 μm, about 1 μm to about 8 μm, about 1 μm to about 5 μm, or about

1 μm to about 2 μm.

In some embodiments, the ball milling is characterised by a speed of about 200 rpm to about 600 rpm for about 30 minutes to about 4 hours. In other embodiments, the speed is about 200 rpm to about 500 rpm, about 200 rpm to about 400 rpm, about 300 rpm to about 600 rpm, about 300 rpm to about 500 rpm, about 300 rpm to about 400 rpm, about 400 rpm to about 600 rpm, or about 400 rpm to about 500 rpm for about 30 minutes to about 3.5 hours, about 30 minutes to about 3 hours, about 30 minutes to about 2.5 hours, about 30 minutes to about 2 hours, about 1 hour to about 4 hours, about 1 hour to about 3.5 hours, about 1 hour to about 3 hours, about 1 hour to about 2.5 hours, about 1 hour to about 2 hours, about 1.5 hours to about 4 hours, about 1.5 hours to about 3.5 hours, about 1.5 hours to about 3 hours, about 1.5 hours to about 2.5 hours, about 1.5 hours to about 2 hours, about 2 hours to about 4 hours, about 2 hours to about 3.5 hours, about 2 hours to about 3 hours, or about 2 hours to about 2.5 hours. In some embodiments, the ball milling is characterised by a speed of about 400 rpm for about 2 hours.

In some embodiments, the phosphoric acid is selected phosphorus oxoacids. Phosphorus oxoacids are acids that consist of phosphorus, oxygen and hydrogen. The phosphorus oxoacids may be orthophosphoric acid (H₃PO₄), pyrophosphoric acid (H4P2O7), hypophosphoric acid (H₄P₂O₆), hypophosphorus acid (H₃PO₂), phosphorus acid (H3PO3), triphosphoric acid (H₅P₃O₁₀), peroxomonophosphoric acid (H₃PO₅), or peroxodiphosphoric acid (H₄P₂O₈). In some embodiments, the phosphoric acid is orthophosphoric acid.

In some embodiments, the phosphoric acid is a phosphoric acid solution characterised by a wt% of about 10 wt% to about 20 wt% of phosphoric acid relative to the phosphoric acid solution. In other embodiments, the wt% is about 10 wt% to about 18 wt%, about 10 wt% to about 16 wt%, about 12 wt% to about 20 wt%, about 12 wt% to about 18 wt%, about 12 wt% to about 16 wt%, about 14 wt% to about 20 wt%, about 14 wt% to about 18 wt%, or about 14 wt% to about 16 wt%. In some embodiments, the wt% is about 16 wt%. In some embodiments, step a) further comprises stirring the calcium carbonate with phosphoric acid at a temperature of about 50 °C to about 100 °C and at a stirring speed of about 200 rpm to about 2000 rpm for about 0.5 hour to about 30 hours. In other embodiments, the temperature is about 50 °C to about 90 °C, about 50 °C to about 80 °C, about 60 °C to about 100 °C, about 60 °C to about 90 °C, about 60 °C to about 80 °C, about 70 °C to about 100 °C, about 70 °C to about 90 °C, about 70 °C to about 80 °C, about 80 °C to about 100 °C, or about 80 °C to about 90 °C. In some embodiments, the temperature is about 80 °C.

In other embodiments, the stirring is performed at a stirring speed of about 200 rpm to about 900 rpm, about 200 rpm to about 800 rpm, about 300 rpm to about 1000 rpm, about 300 rpm to about 900 rpm, about 300 rpm to about 800 rpm, about 400 rpm to about 1000 rpm, about 400 rpm to about 900 rpm, or about 400 rpm to about 800 rpm. In some embodiments, the stirring speed is about 400 rpm to about 800 rpm.

In other embodiments, the stirring is performed for a duration of about 0.5 hour to about 25 hours, about 0.5 hour to about 20 hours, about 0.5 hour to about 15 hours, about 0.5 hour to about 10 hours, about 0.5 hour to about 5 hours, about 1 hour to about 30 hours, about 1 hour to about 25 hours, about 1 hour to about 20 hours, about 1 hour to about 15 hours, about 1 hour to about 10 hours, about 1 hour to about 5 hours, about 10 hours to about 30 hours, about 10 hours to about 25 hours, about 10 hours to about 20 hours, about 10 hours to about 15 hours, about 15 hours to about 30 hours, about 15 hours to about 25 hours, about 15 hours to about 20 hours, about 20 hours to about 30 hours, or about 20 hours to about 25 hours. In some embodiments, the stirring is performed for about 24 hours.

In some embodiments, step a) further comprises stirring the calcium carbonate with phosphoric acid at a temperature of about 80 °C and at a stirring speed of about 400 rpm to about 800 rpm for about 24 hours.

Stirring may allow for homogenisation of mixable liquids and the stir-up of solid particles in liquids. It may also increase the speed of the reaction between calcium carbonate and phosphoric acid. Stirring may be done with a magnetic stirrer or with a mechanical stirrer. In some embodiments, the stirring is mechanical stirring. In some embodiments, step a) further comprises drying of the calcium phosphate powder at a temperature of about 50 °C to about 100 °C for about 8 hours to about 24 hours. In other embodiments, the drying is performed at a temperature of about 50 °C to about 90 °C, about 50 °C to about 80 °C, about 50 °C to about 70 °C, about 60 °C to about 100 °C, about 60 °C to about 90 °C, about 60 °C to about 80 °C, about 60 °C to about 70 °C, about 70 °C to about 100 °C, about 70 °C to about 90 °C, or about 70 °C to about 80 °C. In some embodiments, the drying is performed at a temperature of about 70 °C.

In other embodiments, the duration of drying is about 8 hours to about 20 hours, about 8 hours to about 16 hours, about 8 hours to about 12 hours, about 12 hours to about 24 hours, about 12 hours to about 20 hours, or about 12 hours to about 16 hours. In some embodiments, the duration of drying is about 12 hours.

In some embodiments, step a) further comprises drying of the calcium phosphate powder at a temperature of about 70 °C for about 8 hours to about 24 hours. In some embodiments, step a) further comprises drying of the calcium phosphate powder at a temperature of about 70 °C for about 12 hours.

In some embodiments, the method further comprises a step after a) of ball milling the calcium phosphate powder. The calcium phosphate powder formed from the reaction of calcium carbonate and phosphoric acid may agglomerate and result in an increase in particle size. Ball milling the calcium phosphate powder may break up the agglomerated powder into smaller particles and result in particles with a more uniform size and shape.

In some embodiments, the ball milled calcium phosphate powder is characterised by a particle size distribution with about 35% to about 60% of particles having a particle size of less than about 1 μm. In other embodiments, the particle size distribution is about 35% to about 55%, about 35% to about 50%, about 35% to about 45%, about 35% to about 40%, about 40% to about 60%, about 40% to about 55%, about 40% to about 50%, about 40% to about 45%, about 45% to about 60%, about 45% to about 55%, or about 45% to about 50%. of particles having a particle size of less than about 1 μm. In some embodiments, the calcium carbonate powder is characterised by a particle size distribution with about 54% of particles having a particle size of less than 1 μm. In some embodiments, the method further comprises a step after a) of ball milling the calcium phosphate powder, the calcium phosphate powder characterised by a particle size distribution with about 35% to about 60% of particles having a particle size of less than about 1 μm. In some embodiments, the method further comprises a step after a) of ball milling the calcium phosphate powder, the calcium phosphate powder characterised by a particle size distribution with about 54% of particles having a particle size of less than about 1 μm.

In some embodiments, the calcium phosphate powder is characterised by a particle size of about 0.5 μm to about 5 μm. In other embodiments, the particle size is about 0.5 μm to about 4 μm, about 0.5 μm to about 3 μm, about 0.5 μm to about 2 μm, about 1 μm to about 5 μm, about 1 μm to about 4 μm, about 1 μm to about 3 μm, or about 1 μm to about 2 μm.

In some embodiments, the ball milling is characterised by a speed of about 200 rpm to about 600 rpm for about 30 minutes to about 2 hours. In other embodiments, the speed is about 200 rpm to about 500 rpm, about 200 rpm to about 400 rpm, about 300 rpm to about 600 rpm, about 300 rpm to about 500 rpm, about 300 rpm to about 400 rpm, about 400 rpm to about 600 rpm, or about 400 rpm to about 500 rpm for about 30 minutes to about 3.5 hours, about 30 minutes to about 3 hours, about 30 minutes to about 2.5 hours, about 30 minutes to about 2 hours, about 1 hour to about 4 hours, about 1 hour to about 3.5 hours, about 1 hour to about 3 hours, about 1 hour to about

2.5 hours, about 1 hour to about 2 hours, about 1.5 hours to about 4 hours, about 1.5 hours to about 3.5 hours, about 1.5 hours to about 3 hours, about 1.5 hours to about

2.5 hours, about 1.5 hours to about 2 hours, about 2 hours to about 4 hours, about 2 hours to about 3.5 hours, about 2 hours to about 3 hours, or about 2 hours to about

2.5 hours. In some embodiments, the ball-milling is characterised by a speed of about 400 rpm for about 1 hour.

When additional calcium source is not added, the final mole ratio of calcium phosphorus is provided by the mole ratio of calcium carbonate and phosphoric acid in step a). The excess calcium carbonate remains as unreacted CaCO3 powder, which after sintering, breaks down to CaO, which would absorb water in the environment or when in use and weaken the scaffold. This provides a further degree of control for managing the biodegradability of the calcium phosphate scaffold. In some embodiments, the method further comprises adding additional calcium source to the calcium phosphate powder before the printing and/or sintering step. This may be performed after step a). For example as illustrated in Figure 3, when the mole ratio of calcium to phosphorus in step a) is about 0.5, the resultant calcium phosphate powder is Ca(H₂PO₄)₂'H₂O. By adding additional calcium source at a mole ratio of about 1.2 to about 1.3 to the Ca(H₂PO₄)₂‘H2O powder in step b) before the printing and/or sintering step, a HA product may be formed after sintering. By adding additional calcium source at a mole ratio of about 0.9 to about 1 to the powder before the printing and/or sintering step, a (β-TCP product may be formed after sintering. By adding additional calcium source at a mole ratio of about 1 to about 1.2 to the powder before the printing and/or sintering step, a biphasic calcium phosphate (BCP) product may be formed after sintering.

The calcium source may be calcium carbonate or calcium oxide. In some embodiments, the additional calcium source is calcium carbonate.

When additional calcium source is added, the final mole ratio of calcium to phosphorus is maintained from about 1.3 and about 1.9, or preferably about 1.5 to about 1.7 for the biphasic calcium phosphate product, preferably about 1.4 to about 1.5 for (β-TCP product and preferably about 1.7 to about 1.8 for HA product. In this regard, the mole ratio of calcium to phosphorus in step a) may be lower than about 1.3 to about 1.9 (relatively more phosphorus to calcium) such that the addition of the calcium source in step b) brings the mole ratio to the desired value.

In some embodiments, the mole ratio of calcium to phosphate in step a) is about 1 to about 1.3. In some embodiments, the mole ratio of calcium to phosphate in step a) is about 1.

In some embodiments, the biphasic calcium phosphate product and/or calcium phosphate product is characterised by a calcium phosphate to calcium oxide ratio of about 5: 1 to about 1 : 1. In other embodiments, the ratio is about 5: 1 to about 10:9, about 5: 1 to about 5:4, about 5:1 to about 2: 1, about 5:1 to about 10:3, about 10:3 to about 1: 1, about 10:3 to about 10:9, about 10:3 to about 5:4, about 10:3 to about 2: 1, about 2:1 to about 1 : 1, about 2: 1 to about 10:9, about 2: 1 to about 5:4, about 5:4 to about 1 : 1, or about 5:4 to about 10:9. In some embodiments, the biphasic calcium phosphate product and/or calcium phosphate product is characterised by a calcium phosphate to calcium oxide ratio of about 1.1: 1 to about 2.5: 1. For example, a calcium phosphate to calcium oxide ratio of about 2:1 to about 2.2: 1 may give a (β-TCP product; a ratio of about 1.4:1 to about 2: 1 may give a BCP product; and a ratio of about 1.25: 1 to about 1.4:1 may give a HA product.

Calcium oxide is formed from the decomposition of calcium carbonate during sintering. In use, calcium oxide is dissolvable in water, thus may be removed to allow for cell growth and tissue regeneration. As the rate of biodegradation of calcium oxide, β-TCP and HA differs, by appropriate selection of the ratios, a bone scaffold with tunable biodegradability may be formed, in which the scaffold is gradually degraded to allow for cell growth and tissue regeneration while still providing structural support.

Bio-inks are materials which have been selected for its biocompatibility and favourable rheological properties and may be used for 3D printing. Bio-inks support living cells and may contain living cells and biomaterials that mimic the extracellular matrix environment, supporting cell adhesion, proliferation, and differentiation after printing. Bio-inks may be printed at temperatures that do not exceed physiological temperatures. The bio-ink may be printed via extrusion-based printing, laser-based printing and inkjet printing. Extrusion-based printing entails extruding a continuous stream of viscous liquid through an orifice, such as a nozzle or a syringe. Direct ink writing is an extrusion-based 3D printing technique in which liquid or semisolid colloidal inks are dispensed through small nozzles under controlled pressure and flow rates and may be carried out at room temperature. The bio-ink is extruded from a nozzle head or syringe to form a continuous self-standing filament without spreading, collapsing, or sagging.

In some embodiments, the bio-ink is characterised by a weight percentage of about 30 wt% to 60 wt% of calcium phosphate powder relative to a total composition of the bioink. In other embodiments, the weight percentage is about 30 wt% to about 50 wt%, about 30 wt% to about 40 wt%, about 40 wt% to about 60 wt%, or about 40 wt% to about 50 wt% relative to a total composition of the bio-ink. This range provides for acceptable shrinkage and suitable rheological properties for printing.

In some embodiments, the bio-ink further comprises a solvent. In some embodiments, the solvent is selected from ethyl acetate, acetic acid, ethanol, and water. In some embodiments, the solvent is water.

In some embodiments, the binder is selected from polyvinyl alcohol (PVA), polylactic acid (PLA), poly lactic-co-glycolic acid (PLGA), polycaprolactone (PCL) and a combination thereof. In some embodiments, the binder is polyvinyl alcohol. The calcium phosphate powder may be homogenously dispersed within the binder. This may be done by using high shear mixing.

The bio-ink is a viscoelastic ink possessing shear-thinning behaviour for printing via direct ink writing. Viscoelasticity is the property of a material that exhibits both viscous and elastic characteristics under stress and deformation such as strain. Storage modulus and loss modulus of the bio-ink are parameters that may characterise the bio-ink's behaviour. The storage modulus (G^') represents the bio-ink's ability to store energy and exhibit elastic behaviour. A higher storage modulus may indicate a more elastic and stiffer material. The loss modulus (G") represents the bio-ink's ability to dissipate energy and exhibit viscous behaviour. A higher loss modulus may indicate a more viscous and less elastic material. When the bio-ink is under no or low shear strain, its storage modulus may be higher than its loss modulus where the bio-ink behaves like a gel. The gel-like behaviour may allow the 3D printed structure to be stackable and may support the structural integrity of the 3D printed structure, enabling it to maintain its shape as a 3D scaffold. When the bio-ink is under moderate shear strain or when the shear strain increases, its loss modulus may be higher than its storage modulus. The bio-ink may undergo a transition to a more fluid state and behaves like a liquid, facilitating extrusion of the bio-ink through the nozzle of a 3D printing device.

In some embodiments, the bio-ink is characterised by a storage modulus greater than a loss modulus at a shear strain of less than about 0.5%. In some embodiments, the bio-ink is characterised by a loss modulus greater than a storage modulus at a shear strain of more than 0.5%.

Shear-thinning behaviour is the non-Newtonian behaviour of fluids whose viscosity decreases under shear strain. This allows the bio-ink to become thin and may be easily extruded out during printing. This may be advantageous for direct ink writing printing. This rheology behaviour facilitates ink extrusion under pressure and allows for shape retention after deposition.

In some embodiments, the bio-ink is characterised by a decrease in viscosity with increasing shear rate. In some embodiments, the bio-ink is characterised by a decrease in viscosity from about 10⁴ Pa'S to about 10 Pa'S with increasing shear rate from about 1 s'¹ to about 100 s^-1, the bio-ink with about 46 wt% solid loading. The wt% solid loading for the bio-ink refers to the wt% of calcium phosphate powder present in the bio-ink. This may be the wt% of calcium phosphate relative to a total composition of the bioink. In some embodiments, the bio-ink is characterised by a decrease in viscosity from about 10⁴ Pa'S to about 10 Pa'S with increasing shear rate from about 1 s^-1 to about 100 s’¹, the bio-ink with a wt% of about 46 wt% of calcium phosphate powder relative to a total composition of the bio-ink.

In some embodiments, the 3D printing is direct ink writing. The viscosity range for direct ink writing may be 1 mPa'S to 10 mPa-s.

In some embodiments, the method is characterised by a printing pressure of about 40 kPa to about 400 kPa. In other embodiments, the printing pressure is about 40 kPa to about 350 kPa, about 40 kPa to about 300 kPa, about 50 kPa to about 400 kPa, about 50 kPa to about 350 kPa, or about 50 kPa to about 300 kPa. In some embodiments, the printing pressure is about 50 kPa to about 300 kPa.

In some embodiments, the method is characterised by a printing speed of about 1 mm/s to about 20 mm/s. In other embodiments, the printing speed is about 1 mm/s to about 18 mm/s, about 1 mm/s to about 15 mm/s, about 3 mm/s to about 20 mm/s, about 3 mm/s to about 18 mm/s, about 3 mm/s to about 15 mm/s, about 5 mm/s to about 20 mm/s, about 5 mm/s to about 18 mm/s, or about 5 mm/s to about 15 mm/s. In some embodiments, the printing speed is about 5 mm/s to about 15 mm/s.

Sintering is the process of compacting and forming a solid mass of material by pressure or heat without melting it to the point of liquefaction. Sintering happens as part of a manufacturing process used with metals, ceramics, plastics, and other materials. The particles in the sintered material diffuse across the boundaries of the particles, fusing the particles together and creating a solid piece. Sintering process reduces porosity and enhances properties such as strength and thermal conductivity. During the sintering process, atomic diffusion drives powder surface elimination in different stages, starting at the formation of necks between powders to final elimination of small pores at the end of the process. Sintering may be carefully applied to enhance the strength of a material while preserving porosity. The source of power for solid-state processes is the change in free or chemical potential energy between the neck and the surface of the particle. This energy creates a transfer of material through the fastest means possible; if transfer were to take place from the particle volume or the grain boundary between particles, particle count would decrease and pores would be destroyed. Pore elimination is fastest in samples with many pores of uniform size because the boundary diffusion distance is smallest. Control of temperature is very important to the sintering process, since grainboundary diffusion and volume diffusion rely heavily upon temperature, particle size, particle distribution, material composition, and other properties of the sintering environment itself. The particle size of the powders used for sintering and the distribution of the particle size affects the eventual grain size of the material as well as the porosity and presence of pores in the material. Sintering of the calcium phosphate powder at different temperatures and duration allows for the formation of the different calcium phosphates such as heating the powder at about 1300 °C for the formation of HA and at about 1200 °C for the formation of (β-TCP. Sintering may occur in an oxygen- free environment, nitrogen-free environment or atmospheric environment.

In some embodiments, the sintering step is performed at a heating rate of about 1 °C/min to about 15 °C/min. In other embodiments, the heating rate is about 1 °C/min to about 12 °C/min, about 1 °C/min to about 10 °C/min, about 1 °C/min to about 8 °C/min, about 1 °C/min to about 5 °C/min, about 5 °C/min to about 15 °C/min, about 5 °C/min to about 12 °C/min, about 5 °C/min to about 10 °C/min, or about 5 °C/min to about 8 °C/min. In some embodiments, the heating rate is about 5 °C/min.

In some embodiments, the sintering step is performed at a temperature of about 600 °C to about 1000 °C. In other embodiments, the temperature is about 600 °C to about 900 °C, about 600 °C to about 800 °C, about 700 °C to about 1000 °C, about 700 °C to about 900 °C, about 700 °C to about 800 °C, about 800 °C to about 1000 °C, or about 800 °C to about 900 °C. In some embodiments, the temperature is about 800 °C.

In some embodiments, the temperature is maintained at about 600 °C to about 1000 °C for about 0.5 hour to about 4 hours. In other embodiments, the temperature is maintained at about 600 °C to about 900 °C, about 600 °C to about 800 °C, about 700 °C to about 1000 °C, about 700 °C to about 900 °C, about 700 °C to about 800 °C, about 800 °C to about 1000 °C, or about 800 °C to about 900 °C for about 0.5 hour to about 3 hours, for about 0.5 hour to about 2 hours, for about 1 hour to about 4 hours, for about 1 hour to about 3 hours, or for about 1 hour to about 2 hours. In some embodiments, the temperature is maintained at about 800 °C for about 1 hour to about 2 hours.

In some embodiments, the sintering step is performed at a heating rate of about 1 °C/min to about 15 °C/min to a temperature of about 600 °C to about 1000 °C and maintained for a duration of about 0.5 hour to about 4 hours. In some embodiments, the sintering step is performed at a heating rate of about 5 °C/min to a temperature of about 800 °C and maintained for a duration of about 1 hour to about 4 hours. This allows for the complete decomposition of the binder.

In some embodiments, the sintering step is further performed to a temperature of about 1000 °C to about 1800 °C for about 0.5 hour to about 10 hours. In other embodiments, the sintering step is further performed to a temperature of about 1000 °C to about 1500 °C, about 1000 °C to about 1200 °C, about 1200 °C to about 1800 °C, about 1200 °C to about 1500 °C, or about 1500 °C to about 1800 °C for about 0.5 hour to about 8 hours, for about 0.5 hour to about 6 hours, for about 0.5 hour to about 4 hours, for about 0.5 hour to about 1 hour, or 1 hour to about 10 hours, for about 1 hour to about 8 hours, for about 1 hour to about 6 hours, for about 1 hour to about 4 hours, for about 4 hours to about 10 hours, for about 4 hours to about 8 hours, or for about 4 hours to about 8 hours. In some embodiments, the sintering step is further performed to a temperature of about 1300 °C for about 3 hours to about 5 hours. In some embodiments, the sintering step is further performed to a temperature of about 1200 °C for about 1 hour to about 2 hours.

In some embodiments, the sintering step is performed at a heating rate of about 1 °C/min to about 10 °C/min to a temperature of about 600 °C to about 1000 °C and maintained for a duration of about 1 hour to about 3 hours, and subsequently at a temperature of about 1000 °C to about 1800 °C for a duration of about 0.5 hour to about 10 hours. In some embodiments, the sintering step is performed at a heating rate of about 5 °C/min to a temperature of about 800 °C and maintained for a duration of about 1 hour to about 2 hours, and subsequently at a temperature of about 1300 °C for a duration of about 3 hours to about 5 hours. In some embodiments, the sintering step is performed at a heating rate of about 5 °C/min to a temperature of about 800 °C and maintained for a duration of about 1 hour to about 2 hours, and subsequently at a temperature of about 1200 °C for a duration of about 1 hour to about 2 hours.

In some embodiments, the biphasic calcium phosphate product comprises HA particles and β-TCP particles sintered to each other, the particles characterised by an average particle size of about 0.5 μm to about 5 μm. In other embodiments, the average particle size is about 0.5 μm to about 4 μm, about 0.5 μm to about 3 μm, about 0.5 μm to about

2 μm, about 1 μm to about 5 μm, about 1 μm to about 4 μm, about 1 μm to about 3 μm, or about 0.5 μm to about 2 μm. In some embodiments, the average particle size is about 1 μm to about 3 μm.

Porosity in the biphasic calcium phosphate product affects cell and protein adhesion and growth, and also regulates bone mineral formation. The porosity of calcium phosphate also has an effect on bioactivity. The increase in porosity improves contact with body fluids on the surface area. Thus, dissolution rate is enhanced and the presence of pores on the surface affects protein adsorption. The size and number of pores may impact bone ingrowth and angiogenesis, and affect the mechanical strength and shape of the biphasic calcium phosphate product.

In some embodiments, the biphasic calcium phosphate product and/or calcium phosphate product comprises macropores and/or micropores. Micropores may affect protein adsorption and may also inhibit osteoblastic differentiation. Macropores are generally pores with a pore size greater than 100 μm. Cell growth occurs from the surface of the biphasic calcium phosphate product to the depth of the biphasic calcium phosphate product. Cells may bridged macropores with their long cytoplasmic sprouts that linked to walls and on the micropores. Increasing the size of macroporosity may reduce the number of interconnections to cross and therefore may accelerate cellular colonisation. The size of macropores may be controlled by varying 3D printing parameters such as infill density, filament size and layer height. The size of micropores may be controlled by varying sintering conditions. For example, sintering at higher temperatures may result in a larger pore size and a lower porosity. In some embodiments, the biphasic calcium phosphate product and/or calcium phosphate product is characterised by a macropore size of about 200 μm to about 1500 μm. This allows nutrients to be supplied and blood vessels to grow. In other embodiments, the pore size is about 200 μm to about 1200 μm, about 200 μm to about 1000 μm, about 200 μm to about 800 μm, about 200 μm to about 500 μm, about 300 μm to about 1500 μm, about 300 μm to about 1200 μm, about 300 μm to about 1000 μm, about 300 μm to about 800 μm, or about 300 μm to about 500 μm. In some embodiments, the pore size is about 300 μm to about 500 μm.

In some embodiments, the biphasic calcium phosphate product and/or calcium phosphate product is characterised by a micropore size of about 0.5 μm to about 15 μm. This allows for cell adhesion. In other embodiments, the pore size is about 0.5 μm to about 12 μm, about 0.5 μm to about 12 μm, about 0.5 μm to about 10 μm, about 1 μm to about 15 μm, about 1 μm to about 12 μm, about 1 μm to about 10 μm, about 5 μm to about 15 μm, about 5 μm to about 12 μm, or about 5 μm to about 10 μm. In some embodiments, the pore size is about 1 μm to about 10 μm.

In some embodiments, the biphasic calcium phosphate product and/or calcium phosphate product comprises macropores and/or micropores, the macropores having a macropore size of about 200 μm to about 1500 μm and the micropores having a micropore size of about 0.5 μm to about 15 μm. In some embodiments, the biphasic calcium phosphate product and/or calcium phosphate product comprises macropores and/or micropores, the macropores having a macropore size of about 300 μm to about 500 μm and the micropores having a micropore size of about 1 μm to about 10 μm.

In some embodiments, the biphasic calcium phosphate product and/or calcium phosphate product is characterised by a porosity of about 30% to about 95%. In other embodiments, the porosity is about 30% to about 90%, about 40% to about 95%, about 40% to about 90%, about 50% to about 95%, or about 50% to about 90%. In some embodiments, the porosity is about 50% to about 90%.

In some embodiments, the biphasic calcium phosphate product and/or calcium phosphate product is characterised by a compressive strength of about 2 MPa to about 15 MPa. In other embodiments, the compressive strength is about 2 MPa to about 12 MPa, about 2 MPa to about 10 Mpa, about 3 MPa to about 15 MPa, about 3 MPa to about 12 MPa, about 3 MPa to about 10 MPa, about 5 MPa to about 15 MPa, about 5 MPa to about 12 MPa, about 5 MPa to about 10 MPa, or about 5 MPa to about 8 MPa. In some embodiments, the compressive strength is about 3 MPa to about 11 MPa.

In some embodiments, the biphasic calcium phosphate product and/or calcium phosphate product is characterised by a Young's modulus of about 45 MPa to about 150 MPa. In other embodiments, the Young's modulus is about 45 MPa to about 130 MPa, about 45 MPa to about 100 MPa, about 50 MPa to about 150 MPa, about 50 MPa to about 130 MPa, or about 50 MPa to about 100 MPa. In some embodiments, the Young's modulus is about 51 MPa to about 125 MPa.

In some embodiments, the biphasic calcium phosphate product and/or calcium phosphate product is characterised by a cell viability of about 90% to about 100%. In other embodimnets, the cell viability is about 90% to about 98%, about 90% to about 95%, about 90% to about 92%, about 92% to about 100%, about 92% to about 98%, about 92% to about 95%, about 95% to about 100%, about 95% to about 98%, ot about 98% to about 100%. In some embodiments, the cell viability is greater than about 95%.

The present invention further discloses a biphasic calcium phosphate product and/or calcium phosphate product fabricated according to the method as disclosed herein.

The present disclosure also concerns a biphasic calcium phosphate product comprising: a) hydroxyapatite (HA) particles and beta-tricalcium phosphate (β-TCP) particles sintered to each other, the particles having an average particle size of about 0.5 μm to about 5 μm; wherein the biphasic calcium phosphate product is characterised by micropores having a pore size of about 0.5 μm to about 15 μm; wherein the biphasic calcium phosphate product is characterised by macropores having a pore size of about 200 to about 1500 μm; and wherein the biphasic calcium phosphate product is characterised by a porosity of about 30% to about 95%.

The present disclosure also concerns a calcium phosphate powder; wherein the calcium phosphate powder comprises a shell of calcium phosphate and a core of calcium carbonate; wherein the calcium phosphate powder is characterised by a calcium/phosphorus ratio is about 0.5 to about 1.9; wherein the calcium phosphate powder is characterised by a particle size of about 0.5 μm to about 5 μm; and wherein the calcium is obtained from a naturally occurring source.

The present disclosure also concerns a sintered calcium phosphate powder, comprising: a) hydroxyapatite (HA) particles; and b) beta-tricalcium phosphate (β-TCP) particles; wherein the HA particles and β-TCP particles have an average particle size of about 0.5 μm to about 5 μm.

The present invention also discloses a method of implanting a calcium scaffold in a subject in need thereof, wherein the calcium scaffold comprises a biphasic calcium phosphate product and/or calcium phosphate product as disclosed herein. The method may comprise implanting a biphasic calcium phosphate product as disclosed herein. The biphasic calcium phosphate product and/or calcium phosphate product may be a bone regenerative material that supports bone cell and tissue growth.

In some embodiments, the biphasic calcium phosphate product and/or calcium phosphate product is biodegradable in order to allow regeneration of bone tissue. The rate at which the biphasic calcium phosphate product and/or calcium phosphate product degrades depends on the bone type and the injury sustained by the subject. Using the method as disclosed herein, a biphasic calcium phosphate product and/or calcium phosphate product may be produced such that it is biodegradable within about 5 days to about 30 days in order to allow regeneration of bone tissue.

Examples

The inventors proposed a strategy of converting natural biological calcium carbonate sources into 3D-printed biphasic calcium phosphate scaffolds for bone tissue engineering. These scaffolds contains both hydroxyapatite (HA) and beta-tricalcium phosphate (|β-TCP) phases, which may possess remarkable biocompatibility and osteocond activity for bone tissue repair. The ratio of HA and (β-TCP may be precisely controlled by adjusting the Ca/P ratio in the reaction system, allowing for tunable biodegradation rate for various bone types and age groups. The 3D-printed scaffold contained porous structures with macropores of 412 - 1089 μm and micropores of 5 to 10 μm. Compression test showed that the 3D-printed scaffolds are mechanically comparable to human cancellous bones, with compressive strength of 3.05 to 3.90 MPa and Young's Modulus of 55.2 to 74.7 MPa. Furthermore, the 3D-printed scaffolds demonstrated excellent biocompatibility to preosteoblasts. Hence, the proposed nature- derived biphasic bone scaffolds show great potential in bone tissue engineering, and their excellent tunability in mechanical properties and biodegradation rate make them extremely versatile for applications in different bone types and age groups.

The inventors developed a fabrication method that harnesses natural sources of calcium carbonate, including coral bones, mussel shells, and eggshells, and transforms them into 3D-printed biphasic calcium phosphate scaffolds for bone tissue engineering. These scaffolds offer tunable mechanical stiffness that may match with distinct human bone types, excellent biocompatibility for cell growth, and controllable biodegradability rate to suit diverse bone repair requirements.

Figure 1 illustrates the fabrication process of the nature-derived biphasic bone scaffolds. Coral bones were first boiled in water for 1 hour, followed by 30 minutes of ultrasound cleaning to remove organic residuals and impurities. The purified coral bones were then manually crushed and ball-milled at 400 rμm for 1 hour. Subsequently, the natural CaCOs powder was added to 16 wt% phosphoric acid solution at a Ca/P of 0.5 under vigorous mechanical stirring for 1 hour before it was collected via centrifugation and dried at 70 °C overnight. The dried powder underwent another ball milling process to break down the clumps generated during the reaction. For 3D printing of the bone scaffolds, the bioink is formulated as 5 g of reacted powder, 4.6 g of natural CaCO3 powder, 0.6 g of polyvinyl butyral (PVB), and 6 ml of ethanol as solvent. The PVB binder can be replaced with FDA-approved polymers like PCL for implantation in human body. The ink was then loaded into a 3-mL syringe and printed via direct ink writing (DIW) technique using a BIOX6 bioprinter (CELLINK, Sweden). The printing pressure was set to be 50 kPa and travel speed was 5 mm/s. The printed samples were dried in oven at 50 °C overnight and then sintered in a tube furnace. All samples were first heated to 600 to 800 °C at 5 °C/min and maintained for 2 hours, allowing for the complete decomposition of CaCOs into CaO. This was followed by further heating to 1000-1800 °C for 1 hour and cooled down to room temperature. Compressor gas was used to purge the chamber during the sintering process to remove the polymer fumes.

Coral bones were first boiled in water for 1 hour, followed by 30 minutes of ultrasound cleaning to remove organic residuals and impurities. The purified coral bones were then manually crushed and ball-milled at 400 rμm for 2 hours. Subsequently, the natural coral powder was added to 16 wt% phosphoric acid solution at a Ca/P of 1.3 to 1.67 under vigorous mechanical stirring for 24 hours before it was collected via centrifugation and dried at 70 °C overnight. The dried powder underwent another ball milling process to break down the clumps generated during the reaction. For 3D printing of the bone scaffolds, the bioink is formulated as 5 g of reacted powder, 5ml of 5 wt% PVA solution. The ink was then loaded into a 3-mL syringe and printed via direct ink writing (DIW) technique using a BIOX6 bioprinter (CELLINK, Sweden). The printing pressure was set to be 50 - 300 kPa and travel speed was 5 - 15 mm/s. The printed samples were placed in a humidified chamber to allow gradient drying and then sintered in a muffle furnace. All samples were first heated to 800 °C at 5 °C/min and maintained for 1 hours, allowing for the complete decomposition of PVA binder. This was followed by further heating to 1300 °C for 3 - 5 hour for HA and 1200°C for 1 - 2 hour for TCP and cooled down to room temperature. Compressor gas was used to purge the chamber during the sintering process to remove the polymer fumes.

The size distribution of ball-milled natural CaCOa powder and the reacted powder was characterised through scanning electron microscopy (SEM), as depicted in Figure 2. After ball milling, the size of natural CaCOs powder was reduced to less than 10 μm with approximately 45% of the powder measuring smaller than 1 μm, which is favourable as the small particle size increases the reaction area and may guarantee that the CaCOs is fully reacted with H3PO4. For the reacted powder, the particle size is smaller than 5 μm with around 54% smaller than 1 μm. This particle size distribution is advantageous for the 3D printing process, mitigating the risk of nozzle clogging due to larger particles. Additionally, the fine powder size facilitates the densification process during sintering.

Figure 3a-3d shows the x-ray diffraction analysis (XRD) results of natural coral powder, reacted powder at a Ca/P ratio of 0.5, and sintered scaffolds, respectively. In figure 3a, the XRD pattern of natural coral powder confirmed its composition as aragonite-type calcium carbonate. Upon reaction with excessive amount of phosphoric acid, the CaCC₃ underwent complete conversion to Ca(H₂PO₄)₂'H2O (mono-calcium phosphate monohydrate, MCP-M), as shown in Figure 3b. The Ca/P ratio may be an important factor to consider when synthesizing bioactive ceramics like HA and β-TCP . The reacted MCP-M powder has a low Ca/P ratio of 0.5, whereas the Ca/P ratio of desired HA and (3- TCP is 1.7 and 1.5. Therefore, the inventors proposed adding ball-milled CaCOs powder to the MCP-M before sintering to increase the Ca/P ratio to generate HA and (β-TCP constituents. Figure 3c shows the XRD pattern of sintered ceramic scaffold with a Ca/P ratio of 1.6. The result indicated that the primary crystalline constituents of the sintered scaffold were HA and |β-TCP. When the Ca/P ratio was further elevated to 1.7, the sintered ceramics scaffold contained solely HA. These results demonstrated the potential of precise manipulation of the HA/g-TCP ratio in the sintered scaffold by adjusting the amount of natural CaCOs powder added, allowing the inventors to regulate and control the mechanical properties and biodegradation rate of the scaffolds. Alternatively, the Ca/P ratio may be controlled before reaction by adjusting the CaCC₃ to H3PO4 ratio. Excessive amount of CaCC₃ powder can be added to H3PO4 solution to yield a mixture of Ca₃(PO₄)₂ and CaCC₃, which could be directly sintered into biphasic HA/[β-TCP scaffolds.

Figure 4a-4d shows the x-ray diffraction analysis (XRD) results of natural coral powder, reacted powder, sintered |β-TCP scaffolds, biphasic scaffolds (BCP), and HA scaffolds, respectively. In figure 4a, the XRD pattern of natural coral powder confirmed its composition as aragonite-type calcium carbonate. By varying the Ca/P ratio during reaction, the composition of the product can be precisely controlled. Figure 4b shows the XRD pattern of sintered ceramic scaffolds with a Ca/P ratio of 1.3. The result indicated that the primary crystalline constituents of the sintered scaffold were β-TCP. When the Ca/P ratio was further increased to 1.5 or 1.7, the produced ceramic scaffold contained both HA and (β-TCP, or solely HA. These results demonstrated the potential of precise manipulation of the HA/β-TCP ratio in the sintered scaffold by adjusting the Ca/P ratio during acid reaction, allowing the inventors to regulate and control the mechanical properties and biodegradation rate of the scaffolds.

Figure 5a shows the 3D-printed bone scaffold with a dimension of 16 mm x 16 mm x 2.4 mm (L x W x H). After sintering, the scaffold showed a reduction in size to 13 mm x 13 mm x 2 mm with a volume shrinkage of approximately 18 %. The SEM image in Figure 5c showed that the particles before sintering were densely packed with size ranging from 500 nm to 1 μm. After sintering, the particles bonded with each other and the diameter of HA/β-TCP particles was between 1 μm to 3 μm.

To investigate the influence of microlattice structure on the mechanical properties of the scaffolds, the inventors designed three cylindrical scaffolds with different spacing and filament sizes. As shown in Figure 6a-6c, the filament size of scaffold A and B is 350 μm, while the spacing is 1.25 mm and 1.67 mm respectively. Scaffold B and C share the same spacing of 1.67 mm, while the filament size is 350 μm and 610 μm, respectively. Figure 5d-f shows the optical images of the macropores in scaffold A, B, and C, the macropores size were 528 ± 9 μm, 1176 ± 15 μm, and 901 ± 5 μm, respectively. These macropores may be crucial in bone scaffolds as they may facilitate the infiltration of cells, nutrients, and blood vessel formation, thereby promoting the growth of new bone tissues. Notably, the macropores sizes were smaller than the designed values due to the shrinkage during sintering, which may be countered by enlarging the 3D model before printing. Figure 6g-6i shows the SEM images of the filament surfaces within scaffold A, B, and C. The filament sizes were measured to be 412 ± 9 μm, 506 ± 8 μm, and 1089 ± 13 μm, respectively. Micropores with diameter ranging from 5 μm to 10 μm were observed on the filament surface, which may be essential for the cell attachment and growth.

Figure 7 shows the optical and SEM images of HA and β-TCP . As shown in Figure 7a and 7d, both the sintered HA and β-TCP exhibited macropores structures. These macropores may be crucial in bone scaffolds are they may facilitate the infiltration of cells, nutrients, and blood vessel formation, thereby promoting the growth of new bone tissues. Notably, the macropores sizes were smaller than the designed values due to the shrinkage during sintering, which can be countered by enlarging the 3D model before printing. In addition to the macropores, micropores with diameter ranging from 5 μm to 10 μm were observed on the filament surface, which may be essential for the cell attachment and growth.

In the future, micropore morphology may be controlled by optimizing the binder type, binder particle sizes and sintering conditions to generate interconnected micropore network, allowing for improved nutrient diffusion capabilities and enhanced cell responses. Additionally, certain coral species possesses natural interconnected micropore structure that may be retained after 3D printing and sintering process, creating a hierarchical porous structure in the nature-derived bone scaffold.

The stress-strain curves of the 3D-printed scaffold A, B, and C under uniaxial compression were shown in Figure 8a, and the corresponded compressive stress and Young's modulus were compared in Figure 8b and 8c. The compressive stress of scaffold A, B, and C were 3.90 ± 0.31 MPa, 3.05 ± 0.30 MPa, and 3.24 ± 0.18 MPa, respectively. The corresponding Young's modulus was calculated to be 74.7 ± 2.4 MPa, 55.2 ± 1.1 MPa, and 62.4 ± 2.6 MPa. The stress-strain curves of the 3D-printed HA and [β-TCP scaffolds under uniaxial compression were shown in Figure 9a, and the corresponded compressive stress and Young's modulus were compared in Figure 9b and 9c. The compressive stress of HA and β-TCP scaffolds were 10.34 ± 0.44 MPa and 4.54 ± 0.52 MPa. The corresponding Young's modulus was calculated to be 123.26 ± 7.71 MPa and 51.45 ± 2.21 MPa.

The mechanical properties of the 3D-printed scaffolds were comparable with those of the human cancellous bones and may be controlled by adjusting the porosity of the scaffold to match with specific cases.

Figure lOa-c presents the LIVE/DEAD staining results of MC3T3-E1 preosteoblasts cultured on the 3D-printed scaffolds at day 1, day 2, and day 3. The cells attached and spread on the scaffold surface and remained highly viable with viability of 98.1 ± 1.2 % after three days of culture. Cell counting kit - 8 assay (CCK-8) shown in Figure lOd revealed that the preosteoblasts were able to proliferate normally on the 3D-printed scaffold with no significant difference compared to the cells plated on culture dishes. These results demonstrated that the nature-derived biphasic calcium phosphate scaffold may be highly biocompatible and may be able to support the growth and proliferation of preosteoblasts.

Fig. 11(a) presents the CCK-8 assay results for MC3T3-E1 cells cultured on HA and |3- TCP scaffolds, indicating that the preosteoblasts successfully attached and proliferated on both types of scaffolds. Fig. 11(b) shows the LIVE/DEAD staining results after 14 days of culture, revealing that preosteoblast cells uniformly covered the entire filament surface of both HA and |β-TCP scaffolds, with high cell viability (>95%). These results demonstrate the biocompatibility of the HA and β-TCP scaffolds, supporting their potential to facilitate cellular adhesion, proliferation, and survival in bone tissue engineering applications.

Fig. 12(a) illustrates the viscosity profile of a hydroxyapatite (HA) bioink with approximately 46 wt% solid loading across varying shear rates. The bioink exhibits a typical shear-thinning behavior, characterized by a decrease in viscosity with increasing shear rate, which is advantageous for direct ink writing (DIW) printing. Fig. 12(b) presents the viscoelastic properties of the bioink. At low shear strain, the storage modulus (G^') exceeds the loss modulus (G"), indicating a gel-like behavior that supports the structural integrity of the printed material, enabling it to maintain its shape as a 3D scaffold. Under moderate shear strain, the material undergoes a transition to a more fluid state, facilitating extrusion through the nozzle.

A cylindrical hydroxyapatite (HA) scaffold (6 mm x 8 mm) was implanted in the distal femoral condyle of the left leg, and a (3-tricalcium phosphate ((β-TCP) scaffold in the right leg of a New Zealand rabbit. The rabbit resumed normal movement and activity one week after implantation. Figure 13(a)(b) displays the harvested left and right femoral condyles one month post implantation. The absence of inflammation suggests that the scaffolds are biocompatible and well-tolerated by the host bone tissue. Reconstructed micro-CT images in Fig. 13(c) and (d) reveal that both the HA and (β-TCP scaffolds supported bone regeneration, with new bone tissue forming on the scaffold surfaces and infiltrating the macropores, demonstrating their osteoinductive potential. The HA scaffold remained structurally intact after one month of implantation, while noticeable degradation was observed on the surface of the β-TCP scaffold. These findings align with the known faster biodegradation rate of 0-TCP materials.

In summary, the fabrication method has harnessed the potential of natural calcium carbonate sources, such as coral bones, mussel shells, and eggshells, to create advanced 3D-printed biphasic calcium phosphate scaffolds for bone tissue engineering. These scaffolds exhibit a range of properties crucial for effective bone repair and regeneration. The tunable mechanical stiffness allows for tailoring the scaffold's mechanical characteristics to suit various human bone types, while the biocompatibility supports the growth and proliferation of cells. Moreover, the ability to control the biodegradability rate ensures adaptability to diverse bone repair needs for different age groups.

It will be appreciated that many further modifications and permutations of various aspects of the described embodiments are possible. Accordingly, the described aspects are intended to embrace all such alterations, modifications, and variations that fall within the spirit and scope of the appended claims.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

Throughout this specification and the claims which follow, unless the context requires otherwise, the phrase "consisting essentially of", and variations such as "consists essentially of" will be understood to indicate that the recited element(s) is/are essential i.e. necessary elements of the invention. The phrase allows for the presence of other non-recited elements which do not materially affect the characteristics of the invention but excludes additional unspecified elements which would affect the basic and novel characteristics of the method defined.

The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavor to which this specification relates.

Claims

1. A method of fabricating a biphasic calcium phosphate product, comprising: a) reacting calcium carbonate with phosphoric acid to obtain a calcium phosphate powder; b) combining the calcium phosphate powder with at least one binder and optionally an additional calcium source to obtain a bio-ink; c) 3D printing a calcium phosphate structure using the bio-ink; and d) sintering the calcium phosphate structure to obtain the biphasic calcium phosphate product; wherein the biphasic calcium phosphate product comprises hydroxyapatite (HA) and beta-tricalcium phosphate ((β-TCP); and wherein a final mole ratio of HA to |β-TCP is dependent on a final calcium/phosphorus (Ca/P) ratio of the calcium phosphate powder or the bio-ink.

2. The method according to claim 1, wherein the final mole ratio of calcium to phosphorus is about 1.3 to about 1.9.

3. The method according to claims 1 or 2, wherein the final mole ratio of calcium to phosphorus is about 1.5 to about 1.7.

4. The method according to any one of claims 1 to 3, wherein the final mole ratio of HA to 0-TCP is about 1: 100 to about 100:1.

5. A method of fabricating a calcium phosphate product, comprising: a) reacting calcium carbonate with phosphoric acid to obtain a calcium phosphate powder; b) combining the calcium phosphate powder with at least one binder and optionally an additional calcium source to obtain a bio-ink; c) 3D printing a calcium phosphate structure using the bio-ink; and d) sintering the calcium phosphate structure to obtain the calcium phosphate product; wherein the calcium phosphate product comprises beta-tricalcium phosphate (β-TCP); and wherein a final mole ratio of calcium to phosphorus is about 1.3 to about 1.6.

6. A method of fabricating a calcium phosphate product, comprising: a) reacting calcium carbonate with phosphoric acid to obtain a calcium phosphate powder; b) combining the calcium phosphate powder with at least one binder and optionally an additional calcium source to obtain a bio-ink; c) 3D printing a calcium phosphate structure using the bio-ink; and d) sintering the calcium phosphate structure to obtain the calcium phosphate product; wherein the calcium phosphate product comprises hydroxyapatite (HA); and wherein a final mole ratio of calcium to phosphorus is about 1.6 to about 1.9.

7. The methods according to any one of claims 1 to 6, wherein the calcium carbonate is obtained from a naturally occurring source, preferably coral bones, animal bones, bivalves, eggshells and gastropod shells.

8. The methods according to any one of claims 1 to 7, wherein the method further comprises a step before step a) of ball milling the calcium carbonate, the ball milled calcium carbonate characterised by a particle size distribution with about 30% to about 50% of particles having a particle size of less than about 1 μm.

9. The methods according to any one of claims 1 to 8, wherein the calcium carbonate is characterised by a particle size of about 0.5 μm to about 10 μm.

10. The methods according to any one of claims 1 to 9, wherein the phosphoric acid is selected from phosphorus oxoacids.

11. The methods according to any one of claims 1 to 10, wherein the phosphoric acid is a phosphoric acid solution characterised by a wt% of about 10 wt% to about 20 wt% of phosphoric acid relative to the phosphoric acid solution.

12. The methods according to any one of claims 1 to 11, wherein step a) further comprises stirring the calcium carbonate with phosphoric acid at a temperature of about 50 °C to about 100 °C and at a stirring speed of about 200 rμm to about 1000 rμm for about 0.5 hour to about 30 hours.

13. The methods according to claim 12, wherein the stirring is mechanical stirring.

14. The methods according to any one of claims 1 to 13, wherein step a) further comprises drying of the calcium phosphate powder at a temperature of about 50 °C to about 100 °C for about 8 hours to about 24 hours.

15. The methods according to any one of claims 1 to 14, wherein the method further comprises a step after a) of ball milling the calcium phosphate powder, the ball milled calcium phosphate powder characterised by a particle size distribution with about 35% to about 60% of particles having a particle size of less than about 1 μm.

16. The methods according to any one of claims 1 to 15, wherein the calcium phosphate powder is characterised by a particle size of about 0.5 μm to about 5 μm.

17. The methods according to any one of claims 1 to 16, wherein the method further comprises adding additional calcium source to the calcium phosphate powder after step a), wherein the additional calcium source is calcium carbonate.

18. The methods according to any one of claims 1 to 17, wherein the biphasic calcium phosphate product and/or calcium phosphate product is characterised by a calcium phosphate to calcium oxide ratio of about 1.1 :1 to about 2.5: 1.

19. The methods according to any one of claims 1 to 18, wherein the bio-ink is characterised by a wt% of about 30 wt% to 60 wt% of calcium phosphate powder relative to a total composition of the bio-ink.

20. The methods according to any one of claims 1 to 19, wherein the bio-ink further comprises a solvent, the solvent selected from ethyl acetate, acetic acid, ethanol and water.

21. The methods according to any one of claims 1 to 20, wherein the binder is selected from polyvinyl alcohol (PVA), polylactic acid (PLA), poly lactic-co-glycolic acid (PLGA), polycaprolactone (PCL), and a combination thereof.

22. The methods according to any one of claims 1 to 21, wherein the bio-ink is characterised by a storage modulus greater than a loss modulus at a shear strain of less than about 0.5% and a loss modulus greater than a storage modulus at a shear strain of more than 0.5%.

23. The methods according to any one of claims 1 to 22, wherein the bio-ink is characterised by a shear-thinning behaviour.

24. The methods according to any one of claims 1 to 23, wherein the 3D printing is direct ink writing.

25. The methods according to any one of claims 1 to 24, wherein the sintering step is performed at a heating rate of about 1 °C/min to about 10 °C/min to a temperature of about 600 °C to about 1000 °C and maintained for a duration of about 0.5 hour to about 4 hours, and subsequently at a temperature of about 1000 °C to about 1800 °C for a duration of about 0.5 hour to about 10 hours.

26. The method according to any one of claims 1 to 4, 7 to 25, wherein the biphasic calcium phosphate product comprises HA particles and (β-TCP particles sintered to each other, the particles characterised by an average particle size of about 0.5 μm to about 5 μm, preferably about 1 μm to about 3 μm.

27. The methods according to any one of claims 1 to 26, wherein the biphasic calcium phosphate product and/or calcium phosphate product comprises macropores and/or micropores, the macropores having a macropore size of about 200 μm to about 1500 μm and the micropores having a micropore size of about 0.5 μm to about 15 μm.

28. The method according to any one of claims 1 to 27, wherein the biphasic calcium phosphate product and/or calcium phosphate product is characterised by a porosity of about 30% to about 95%.

29. The methods according to any one of claims 1 to 28, wherein the biphasic calcium phosphate product and/or calcium phosphate product is characterised by a compressive strength of about 2 MPa to about 15 MPa.

30. The methods according to any one of claims 1 to 29, wherein the biphasic calcium phosphate product and/or calcium phosphate product is characterised by a Young's modulus of about 45 MPa to about 150 MPa.

31. The methods according to any one of claims 1 to 30, wherein biphasic calcium phosphate product and/or calcium phosphate product is characterised by a cell viability of about 90% to about 100%.

32. A biphasic calcium phosphate product and/or calcium phosphate product fabricated according to any one of claims 1 to 31.

33. A biphasic calcium phosphate product comprising: a) hydroxyapatite (HA) particles and beta-tricalcium phosphate (β-TCP) particles sintered to each other, the particles having an average particle size of about 0.5 μm to about 5 μm; wherein the biphasic calcium phosphate product is characterised by micropores having a pore size of about 0.5 μm to about 15 μm; wherein the biphasic calcium phosphate product is characterised by macropores having a pore size of about 200 μm to about 1500 μm; and wherein the biphasic calcium phosphate product is characterised by a porosity of about 30% to about 95%.

34. A calcium phosphate powder, wherein the calcium phosphate powder is characterised by a calcium/phosphorus ratio is about 0.5 to about 1.9; wherein the calcium phosphate powder is characterised by a particle size of about 0.5 μm to about 5 μm; and wherein the calcium is obtained from a naturally occurring source.

35. A bio-ink, comprising: a) calcium phosphate powder; and b) a binder; wherein the calcium phosphate powder is characterised by a calcium/phosphorus ratio is about 1.3 to about 1.9; and wherein the bio-ink is characterised by a wt% of about 30 wt% to 60 wt% of calcium phosphate powder relative to a total composition of the bio-ink.

36. A method of implanting a calcium scaffold in a subject in need thereof, wherein the calcium scaffold comprises a biphasic calcium phosphate product and/or calcium phosphate product according to any one of claims 1 to 33.

37. The method according to claim 36, wherein the biphasic calcium phosphate product and/or calcium phosphate product is biodegradable in order to allow regeneration of bone tissue.