WO2019232318A1

WO2019232318A1 - Biomimetic 3d printing of hierarchical and interconnected porous hydroxyapatite bone structure

Info

Publication number: WO2019232318A1
Application number: PCT/US2019/034825
Authority: WO
Inventors: Dong Lin; Shuting LEI; Xiaolei Song; Guang Yang
Original assignee: Kansas State University Research Foundation
Priority date: 2018-06-01
Filing date: 2019-05-31
Publication date: 2019-12-05

Abstract

Methods and apparatus are described herein for the production of porous hydroxyapatite tissue scaffold structures. The structures can be formed by a combination extrusion and cast freezing additive manufacture method, similar to a 3D printing process, using a pseudoplastic hydroxyapatite material as the print medium. The resulting structures exhibit hierarchical and interconnected porous structures, which are particularly suitable for bone cell culture.

Description

BIOMIMETIC 3D PRINTING OF HIERARCHICAL AND INTERCONNECTED POROUS

HYDROXYAPATITE BONE STRUCTURE

CROSS-REFERENCE TO RELATED APPLICATIONS The present application claims the priority benefit of U.S. Provisional Application No.

62/679,155, filed June 1, 2018, entitled BIOMIMETIC 3D PRINTING OF HIERARCHICAL AND INTERCONNECTED POROUS HYDROXYAPATITE BONE STRUCTURE, incorporated by reference in its entirety herein. BACKGROUND OF THE INVENTION

Field of the Invention

The present invention is generally directed to a method of forming porous hydroxyapatite bone scaffold structures from an aqueous hydroxyapatite suspension. In specific embodiments, a simultaneous extrusion and unidirectional freezing method is used to fabricate hierarchical and interconnected porous structures for bone cell culture.

Description of Related Art

Bone grafting is a surgical procedure carried out to fix fractured bones and bone injury. The procedure involves transplantation of bone tissues to augment and regenerate bones that are lost due to diseases or injuries. Autografts have been used for decades for the grafting purposes. Autografting is one of the most used available surgical procedures for the bone grafting. However, due to the double incision required during the autograft procedure, the pain associated with the procedure, and the post-operative pain on both the incision sites, there has been a need for alternatives. Thus, the use of the allografts, xenografts, and synthetic bone grafts is on the rise. With the development of these alternatives, the global market for bone graft substitutes is growing.

Increased use of bone grafting procedure to fix damaged/broken bones, high demand of the bone graft substitutes in the military medical units, rising geriatric population with increasing orthopaedic problems, and increased need for the reconstructive orthopaedic treatments has driven the growth of the market. Additionally, increased usage of the bone graft substitutes in the field of dentistry and technological advancements from the leading market players with innovative bone graft products add fuel to the market growth.

Prior art freeze casting methods have difficulty controlling the microstructure due to the complex nature of ice crystallization, and the 3D shape of the freeze casting obj ect is limited. What is needed is a method capable of customizing the macro structure of the object and controlling the formation of interconnected pores that are suitable for human cell culturing.

SUMMARY OF THE INVENTION

The present disclosure is broadly concerned with methods of producing a porous hydroxyapatite structure. The methods generally comprise providing a pseudoplastic material comprising hydroxyapatite dispersed in an aqueous suspension. A first layer of the pseudoplastic material is then deposited onto a surface. The first layer is then cooled to a sufficient temperature so as to freeze the water within the first layer of the pseudoplastic material. A second layer of the pseudoplastic material is then deposited onto the first layer.

Also described herein are porous hydroxyapatite structures formed by the methods according to one or more embodiments of the invention. In one or more embodiments, the structures comprise a hydroxyapatite material and a plurality of interconnected macropores and micropores. The plurality of macropores have a maximum cross-sectional dimension of about 0.1 to about 5 mm. The plurality of micropores have a minimum cross-sectional dimension of about 1 pm to about 50 pm.

Methods of growing cells in vitro are also described herein. The methods generally comprise adding a quantity of cells and tissue culture medium to a porous hydroxyapatite structure according to one or more embodiments of the invention. In one or more embodiments, the methods further comprise introducing a porous hydroxyapatite structure described above into the body of a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure (Fig.) 1 is a schematic of the 3D printing process combining freeze casting and extrusion in accordance with one or more embodiments of the present invention;

Fig. 2 is a series of photographs showing the manufacturing process and samples prepared in accordance with methods of the present invention having different macro structures (scale bar in (d) is lcm);

Fig. 3 is a series of photographs showing the rheology behavior of the hydroxyapatite suspension in accordance with embodiments of the invention;

Fig. 4A is a graph showing shear stress vs. shear rate testing of the suspension of Fig. 3; Fig. 4B is a graph showing viscosity vs. shear rate testing of the suspension of Fig. 3;

Fig. 4C is a graph showing the moduli measurements of the suspension of Fig. 3;

Fig. 5 is a series of photographs showing 3D printed hierarchical and porous hydroxyapatite structures in accordance with embodiments of the invention;

Fig. 6 is a series of photographs showing the surface morphology of 3D printed HA structures in accordance with embodiments of the invention;

Fig. 7 is a pair of photographs showing the shapes of the mesopores being tailored by freezing conditions in accordance with embodiments of the invention, wherein the sample shown was frozen from an initial temperature of -18 °C and then decreased at a rate of -1 °C/min;

Fig. 8 is a series of photographs showing the cross-sectional view of interconnected micropores showing lamella shape in accordance with embodiments of the invention;

Fig. 9 is a series of photographs showing laminar HA wall structures in the cross-section in accordance with embodiments of the invention;

Fig. 10 is a graph and schematic showing the results of an in-plane compression test;

Fig. 11 is a series of photographs showing the results of the in-plane compression test of Fig. 10;

Fig. 12 is a pair of photographs showing the structural integrity of 3D printed HA after sintering in accordance with embodiments of the invention, with particle bonding after sintering at 1300 °C for 3 hours;

Fig. 13 is a series of photographs showing surface morphology of a scaffold in accordance with embodiments of the invention after cell growth;

Fig. 14 is a series of photographs showing cell growth on a top surface of a scaffold in accordance with embodiments of the invention;

Fig. 15 is a series of photographs showing cell growth and migration in the cross-section of printed HAP structures in accordance with embodiments of the invention, cross-section demonstrating thru-growth of cells;

Fig. 16 is a series of photographs showing cell growth and migration in the cross-section of printed HAP structures in accordance with embodiments of the invention;

Fig. 17 is a series of photographs showing cell growth and migration in the cross-section of printed HA structures in accordance with embodiments of the invention;

Fig. 18 is a series of photographs showing cell growth and migration in the cross-section of printed HA structures in accordance with embodiments of the invention; and Fig. 19 is a series of photographs showing the bottom surface morphology of a scaffold in accordance with embodiments of the invention after cell growth.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Embodiments of the present invention are directed to methods and apparatus for producing a porous hydroxyapatite (HAP) structure using a combination of freeze casting and 3D printing techniques. The HAP structure is generally produced from a pseudoplastic material comprising hydroxyapatite dispersed in an aqueous suspension or solvent system and using an additive manufacturing method. The HAP structure is particularly useful as a biocompatible tissue scaffold for bone tissue engineering applications. As used herein,“pseudoplastic” means a material that exhibits the non-Newtonian behavior of fluids whose viscosity decreases under shear strain (i.e., shear thinning).

In one or more embodiments, the method comprises depositing a first layer of the pseudoplastic material onto a surface. In one or more embodiments, the material is extruded, dispensed or otherwise deposited onto the surface using a suitable apparatus that generally includes a chamber for holding the material, with the chamber in fluid communication with a material passage or barrel that terminates in a dispensing tip or outlet. The dispensing tip or nozzle will have an orifice though which the pseudoplastic material is expelled/extruded/dispensed. The technique can be executed using a simple apparatus, such as a syringe with a nozzle, as well as machines specifically designed for extrusion and deposition of the layer. In certain preferred embodiments, the pseudoplastic material is deposited by extrusion of the material, for example by pushing the pseudoplastic using pressure through a nozzle typical of a 3D printing system. The pseudoplastic material may be deposited onto the surface in any number of geometries and having any number of patterns, which may be selected depending on the particular tissue scaffold application. In certain embodiments, the first layer is deposited having a thickness of about 0.01 mm to about 2 mm, preferably about 0.1 mm to about 1 mm. In certain embodiments, the first layer has a pattern comprising a plurality of macropores formed therein, for example as shown in Fig. 2. The macropores may be substantially circular, or may have other geometries such as triangular, rectangular, diamond, etc. Regardless the geometry, in certain embodiments the macropores have an average maximum width of about 0.1 mm to about 10 mm, preferably about 1 mm to about 5 mm, wherein the average maximum width is the mean average length of the widest cross-sectional distance/dimension across each pore. After the first layer is deposited onto the surface, in certain embodiments the method comprises cooling the first layer to a temperature sufficient to freeze the water within the pseudoplastic material. A number of different approaches can be used to achieve or effect cooling of the material. In certain preferred embodiments, the surface upon which the first layer is deposited is integrated with or positioned adjacent to or on top of a cold plate, for example as shown in Fig. 1. In such embodiments, the cooling for the first layer is provided at the interface of the first layer and the surface. It has been discovered that providing cooling in this manner has certain advantages, particularly for bone tissue engineering applications. When the first layer of material is deposited onto the surface, the water phase of the pseudoplastic material is slowly cooled, preferably uniformly, in all directions, resulting in randomly distributed and shaped micropores with irregular crystallization. This irregular crystallization may have a height within the first layer of up to only about 50pm as measured from the surface onto which the first layer is deposited. As the cooling continues, the ice crystals forming in the first layer reach a stable zone, where ice dendrites grow uni directionally towards the temperature gradient (i.e., away from the cold surface).

Once the first layer has been sufficiently cooled (e.g., such that the water phase, and preferably the entire layer, has frozen), the first layer can act as a surface upon which to deposit one or more additional layers of pseudoplastic material. Thus, in certain embodiments the method comprises depositing a second layer of the pseudoplastic material onto the first layer. Similar to the first layer, the second layer of pseudoplastic material may be deposited by extrusion and may be deposited onto the first layer in any number of geometries and having any number of patterns. Also similar to the first layer, the second layer may be deposited having a thickness of about 0.01 mm to about 2 mm, preferably about 0.1 mm to about 1 mm. In certain embodiments, the second layer has a pattern comprising a plurality of macropores formed therein, which may be substantially circular, or may have other geometries such as triangular, rectangular, diamond, etc. In certain embodiments the macropores of the second layer have an average maximum width of about 0.1 mm to about 10 mm, preferably about 1 mm to about 5 mm, wherein the average maximum width is the mean average length of the widest cross-sectional distance across each pore. In certain preferred embodiments, the second layer is deposited having a pattern such that the plurality of macropores of the second layer align with the plurality of macropores of the first layer to define a plurality of aligned macropore channels passing through the both first and second layers. However, it is within the scope of the present invention that the first and second layers may have the same or different patterns from each other and the same or different patterns shown in the figures herein.

After the second layer is deposited onto the first layer, in certain embodiments the method comprises cooling the second layer to a temperature sufficient to freeze the water within the pseudoplastic material. Similar to the first layer, a variety of approaches can be used to effect or achieve cooling of the second layer. However, when the surface upon which the first layer is deposited is integrated with or positioned adjacent to or on top of a cold plate, the temperature of the cold plate is preferably low enough to provide sufficient cooling through the first layer and to the second layer to freeze the water within the pseudoplastic material of the second layer. Alternatively, the temperature of the cold plate can be constantly or periodically lowered to provide increased cooling to freeze the water within the pseudoplastic material of the second layer. Similar to the first layer, the water phase of the pseudoplastic material in the second layer can be slowly cooled, preferably uniformly, in all directions, resulting in randomly distributed and shaped micropores with irregular crystallization, followed by the formation of unidirectional ice dendrites away from the cold surface.

In certain embodiments, one or more additional layers of the pseudoplastic material may be deposited and cooled on top of the second layer using the same or different methods as described above with respect to the first and second layers. Each of the additional layers may have the same or different patterns from the first and second layers described above. Additionally, each of the additional layers may have the same or different thickness, as well as the same or different macropore arrangement and dimensions as the first and second layers described above. In certain preferred embodiments, the additional layers are deposited having a pattern such that the plurality of macropores of the all layers align to define a plurality of aligned macropore channels passing through the scaffold structure. The total number of layers depends on the desired height of the scaffold. Similarly, the length and width of the first, second, and any additional layers depends on the desired length and width of the scaffold product. It has been discovered that micropore patterns within the final scaffold can be adjusted depending on the particular freezing conditions of the ice within the pseudoplastic material, including starting temperature and cooling rate. Thus, the temperatures applied to the deposited layers can be selected and altered to achieve the desired micropore pattern. However, in certain embodiments, during the depositing of the first layer of pseudoplastic material, the cold plate begins at a temperature of about -40 °C to about 10 °C, preferably about -20 °C to about 5 °C. In certain embodiments, the temperature of the cold plate is decreased during formation of subsequent layers at a rate of about 0.1 °C to about 5 °C per minute, and preferably about 0. 5 °C to about 2 °C per minute.

After all layers of the pseudoplastic material have been deposited, in certain embodiments the method comprises freezing the resulting layered structure formed by the layers at a temperature cold enough to provide thermal equilibrium to all layers and to form a solid intermediate structure comprising a solid ice phase and a solid hydroxyapatite phase. In certain embodiments, the solid intermediate structure is a single, monolithic body. However, in certain other embodiments, the layers may still be separable at this step in the process. The layered structure may remain attached to the surface during this freezing step, or the layered structure may be removed from the depositing surface prior to freezing. The freezing time and temperatures may vary, so long as thermal equilibrium is achieved with defined (and separate) ice and hydroxyapatite phases. In certain embodiments, the layers are subjected to a temperature of about -120 °C to about -25 °C, preferably about -50 °C to about -90 °C, for a time of about 1 hour to about 24 hours, preferably about 8 hours to about 16 hours. After the intermediate structure is sufficiently frozen, the method comprises freeze drying the solid intermediate structure, for example using known freeze dry methods, thereby removing the ice phase from the solid intermediate structure. The removal of the ice phase crystals and dendrites from the layers of the intermediate structure reveals an interconnected network of pores (micropores) within the solid intermediate structure. After the ice phase is removed, the method comprises heating the solid intermediate structure to a temperature sufficient to sinter the hydroxyapatite phase. In certain embodiments, the structure is subjected to a temperature of about 500 °C to about 2000 °C, preferably about 1000 °C to about 1000 °C, for a time of about 20 minutes to about 10 hours, preferably about 1 hour to about 5 hours. The sintering step results in a porous hydroxyapatite (HAP) structure product, preferably in monolithic form with the layers being inseparable.

In certain embodiments, the methods of the present invention are performed using an apparatus configured for simultaneous extrusion of the pseudoplastic material and cast freezing. In certain such embodiments, the apparatus may comprise equipment and configurations similar to a 3D printer. In certain embodiments, the first, second, and/or additional layers of the pseudoplastic material is deposited by extruding the pseudoplastic material through a nozzle. The surface upon which the first layer is deposited may comprise any of a number of materials that are useful, for example as 3D printer bed materials. However, in certain embodiments, the surface material should be selected based on the thermal conductivity so as to control the cooling and freezing of the water within the layers of pseudoplastic material, as described above. In certain embodiments, the surface comprises a material selected from the group consisting of glass, acrylic, and metal. In certain embodiments, the surface comprises a coating, which will assist in the removal of the scaffold from the surface at the desired point in the fabrication process. In certain embodiments, cooling is provided to the layers during fabrication via indirect heat exchange with a cooling fluid introduced into a cold box below the printing surface. In certain such embodiments, the cooling fluid is liquid nitrogen, although other fluids may be used within the scope of the present invention.

The pseudoplastic material used in the methods of the present invention generally comprises hydroxyapatite dispersed in an aqueous suspension or solvent system. In certain embodiments, wherein the pseudoplastic material comprises from about 40% to about 80% by weight, preferably about 50% to about 70% by weight of hydroxyapatite, with the total weight of the material taken as 100% by weight. Preferred aqueous solvent systems include water (and may consist essentially or even consist of water), including tap water or purified water, including distilled water, deionized water, demineralized water, and the like. In certain embodiments, the material is a suspension of hydroxyapatite powder dispersed in water (as the aqueous solvent system). In certain such embodiments, a dispersant may be added to the suspension. In certain embodiments, the dispersant is included at a level of about 0.1% to about 5% by weight, preferably about 0.5% to about 2% by weight, based on the total weight of hydroxyapatite powder taken as 100% by weight. An exemplary dispersant is ammonium polymethacrylate. In certain embodiments, a viscosifier may be added to the suspension. In certain embodiments, the viscosifier is added to the suspension at a level of about 0.1% (w/v) to about 10% (w/v), preferably about 0.5% (w/v) to about 5% (w/v). An exemplary viscosifier is hydroxypropyl methyl cellulose. Other additives may also be included as desired, such as defoamers, pH adjusting agents, surfactants, and the like. An exemplary defoamer is l-octonal. Exemplary pH adjusting agents are nitric acid and sodium hydroxide. In certain embodiments, the suspension has a pH of about 8 to about 10. Finally, a gelling agent may be added to suspension. In certain embodiments, the gelling agent is included at a level of about 0.2% to about 10% by weight, preferably about 1% to about 3% by weight, based on the total weight of hydroxyapatite powder taken as 100% by weight. An exemplary gelling agent is polyethylenimine.

Pseudoplastic materials described herein are particularly suitable for both extrusion printing and freeze casting. To achieve this, the pseudoplastic materials should be sufficiently viscoelastic to keep its shape when deposited while also being sufficiently flowable so that the hydroxyapatite particles inside can be squeezed during ice crystallization. Thus, the pseudoplastic materials described herein advantageously exhibit shear thinning pseudoplastic properties. In certain embodiments, the deposited pseudoplastic material is self-sustaining. As used herein, the term“self-sustaining” means that the material retains its shape without an external support structure or container, and is not susceptible to deformation merely due to its own internal forces. The self-sustaining material is also flowable, like a gel, and will not recoil or spring back into shape after stretching and/or compression. Thus, when an external pressure or force is applied to the self-sustaining material, it can be shaped or extruded. Thus, the flowable pseudoplastic material can be drawn into and/or dispensed from a nozzle. In certain embodiments, the pseudoplastic material has a shear stress of about 50 Pa to about 150 Pa, preferably about 75 Pa to about 125 Pa, when the shear rate is zero at room temperature. In certain embodiments, the pseudoplastic material has a minimum viscosity of about 0.5 Pa-s to about 2 Pa-s, preferably about 0.7 Pa-s to about 1 Pa-s, at a shear rate of 100 s ¹ at room temperature. In certain embodiments, when subjected to a shear stress of about 75 Pa to about 200 Pa, the storage modulus is about 25 Pa to about 50 Pa, and the loss modulus is about 30 Pa to about 120 Pa, respectively.

Porous hydroxyapatite structures formed in accordance with embodiments of the present invention are particularly suitable for use as tissue scaffold for bone tissue applications. This is due to the hierarchical porous network as well as the compressive strength of the structures. In certain embodiments, the porous hydroxyapatite structure comprises a plurality of interconnected macropores and micropores. In certain embodiments, the plurality of macropores have a maximum cross-section of about 0.1 to about 5 mm, preferably about 0.4 mm to about 2 mm. In certain embodiments, the plurality of micropores have a minimum cross-section of about 1 pm to about 50 pm, preferably about 5 pm to about 10 pm. The compressive strength of the hierarchical porous structure is generally anisotropic. In certain embodiments, the porous hydroxyapatite structure has a compressive strength of at least about 10 MPa, preferably at least about 20 MPa, in parallel to the freeze casting direction. In certain embodiments, the porous hydroxyapatite structure has a compressive strength of at least about 1 MPa, preferably at least about 2 MPa, perpendicular to the freeze casting direction.

The methods, apparatus, and products in accordance with embodiments of the present invention have a number of benefits and advantages. The porous hydroxyapatite scaffold structure successfully mimics the hierarchical human bone structure, having interconnected pores that range from few micrometers to a centimeter. The scaffold is also biocompatible and permits cell adhesion and migration. As used herein, the term“biocompatible” means the scaffold material does not have toxic or injurious effects on biological systems, particularly within the human body. The interconnected micropores provide channels for the cells, such as mesenchymal cells, stem cells, osteoblasts, chondrocytes, and the like, to grow into or invade the scaffold and greatly enhance the cell growth sites. The printing methods and suspensions described herein provide a high weight ratio of hydroxyapatite material, which eliminates the boundaries between layers, thus demonstrating high compressive strength for real applications (demonstrating higher compressive strength compared to prior art scaffolds). The superior mechanical properties and cell culture performance demonstrate the great potential of methods for bone tissue engineering applications. Moreover, the 3D printing aspect allows for ease of manufacturing macrostructures with controlled dimensions. Using simultaneous extrusion and unidirectional freezing method to fabricate hierarchical and interconnected porous hydroxyapatite structures for bone cell culture. Thus, the porous hydroxyapatite structure is a feasible synthetic substitute for autografts, and for use in spinal fusion, long bone, joint reconstruction, foot and ankle, dental, and other procedures.

Additional advantages of the various embodiments of the invention will be apparent to those skilled in the art upon review of the disclosure herein and the working examples below. It will be appreciated that the various embodiments described herein are not necessarily mutually exclusive unless otherwise indicated herein. For example, a feature described or depicted in one embodiment may also be included in other embodiments, but is not necessarily included. Thus, the present invention encompasses a variety of combinations and/or integrations of the specific embodiments described herein.

As used herein, the phrase“and/or,” when used in a list of two or more items, means that any one of the listed items can be employed by itself or any combination of two or more of the listed items can be employed. For example, if a composition is described as containing or excluding components A, B, and/or C, the composition can contain or exclude A alone; B alone; C alone; A and B in combination; A and C in combination; B and C in combination; or A, B, and C in combination.

The present description also uses numerical ranges to quantify certain parameters relating to various embodiments of the invention. It should be understood that when numerical ranges are provided, such ranges are to be construed as providing literal support for claim limitations that only recite the lower value of the range as well as claim limitations that only recite the upper value of the range. For example, a disclosed numerical range of about 10 to about 100 provides literal support for a claim reciting“greater than or equal to about 10” (with no upper bounds) and a claim reciting“less than or equal to about 100” (with no lower bounds).

EXAMPLE

The following example sets forth the preparation of pseudoplastic hydroxyapatite materials, a method of processing the materials to form a porous hydroxyapatite structure, and testing of the structure, in accordance with embodiments of the present invention. It is to be understood, however, that this example is provided by way of illustration and nothing therein should be taken as a limitation upon the overall scope of the invention.

Experimental

Suspension preparation: The suspension was prepared by mixing commercial hydroxyapatite powders (dso=l-3 micrometer, Trans-Tech, Adamstown, MD) in deionized (DI) water at a concentration of 60 wt.%. Darvan C-N (Vanderbilt Minerals, Norwalk, CT) was added by 0.8 wt.% based on the hydroxyapatite solid to disperse the hydroxyapatite powder. The suspension was mixed homogenously for 5 hours by magnetic stirring. Thereafter hydroxypropyl methyl cellulose (H7509, Sigma-Aldrich, Saint Louis, MO), as a viscosifier, was added by 9 mg/mL in the DI water, followed by stirring for 12 hours for full dissolving. During this step, 1- octonal (112615, Sigma-Aldrich, Saint Louis, MO) was also added by 0.5 vol.% of the distilled water as a defoamer. Finally, Polyethylenimine (PEI, Mw~l800, Polyscience, Warrington, PA) was added by 1.6 wt.% of the powder to trigger the gelling process, also stirred for 12 hours. After each step, the pH value of the suspension was adjusted to 9 using concentrated nitric acid or sodium hydroxide.

Rheology testing: The suspension rheology was characterized using an ARl500ex rheometer (TA Instruments, New Castle, DE) with a 25 mm parallel plate geometry and a gap of 50 pm. All measurements were performed by sweeping the strain amplitude at a constant angular frequency of 1 Hz and a constant temperature of 5 °C.

Extrusion: The extrusion was completed on a commercial fused deposition modeling (FDM) 3D printer (Airwolf 3D HD2x, Costa Mesa, CA) modified by replacing fusing modulus with a customized holder. The HAP suspension-loaded syringe was mounted on the holder so that its movement can be controlled by the printer. The air pressure was controlled by a high precision dispenser (Ultimus V, Nordson EFD, East Providence, RI) for a continuous flow of the suspension. The extruded suspension was deposited on a hot/cold plate (TP294, Sigma Systems, El Cajon, CA) with controlled temperature, where the initial temperature was 5°C and then decreased by l°C/min after the extrusion.

Post-processing: The frozen samples were kept in a freezer at -70°C for 12 hours for further freezing. Next, the samples were sublimated by a FreeZone Trial freeze dry system (Labconco, Kansas City, MO). Finally, the dried samples were transferred to a furnace (Kejia, Zhengzhou, China), where the temperature was ramped up to 500°C and kept there for 2 hours to remove the polymers, and then raised to l300°C and kept for 3 hours to sinter the samples, both with a ramp rate of 2°C/min.

Scanning electron microscopy (SEM) characterization: For preparation, the samples were sputter-coated with gold. SEM images were taken at 3kV on a FEI Helios NanoLab 660 (FEI, Hillsboro, OR).

Mechanical testing: The final samples were cut into small cubes and polished by sandpaper. Then the cubes were compressed at a speed of lmm/min on an EZ Test table-top tester (Shimadzu, Kyoto, Japan).

Cell culturing: Human mesenchymal stromal cells were prepared and characterized using the methods known in the art. Briefly, discarded human umbilical cords were obtained from anonymous donors and enzymatically and mechanically dissociated to single cells and placed into primary culture with cell culture medium consisting of low glucose Dulbecco’s Modified Eagle Medium enriched with 10% pooled human platelet lysate and 1% antibiotic/ antimycotic. Upon expansion, the cells were lifted and replated (called passage). At passage 3, cells were characterized as mesenchymal stromal cells (MSCs) using the International Society of Cellular Therapy’s minimal MSC definition and cell aliquots were stored frozen until use. To load MSCs on scaffolds, the MSCs were thawed and recovered one passage prior to lifting for passaging and seeding onto scaffolds by adding 100,000 cells per well into 6 well plates containing scaffold. The loaded scaffolds were placed back into the incubator and photographed daily using an Evos Auto 2000 microscope. After four or five days of attachment/ growth of MSCs on the scaffold, the medium was replaced with freshly prepared 4% paraformaldehyde in phosphate buffered saline (pH 7.4) for 20-30 min at room temperature to fix the cells in situ. The fixative was removed by 2 rinses with phosphate buffered saline and the plates were stored at 4°C until the scaffolds were prepared for scanning electron microscopy. Results and Discussion

Natural materials, including bone, teeth, bamboo, and wood, demonstrate mechanical superior properties due to their sophisticated hierarchical architecture spanning from the nano/microscopic level to the macroscopic. The hierarchical structure of bone contributes to its superior mechanical behaviors: the outer layer of dense cortical bone with tube-shaped canals of 5-l0pm diameter (microscopic) provides the main compressive strength and the inner cancellous bone with interconnected pores sized from 450 to l3 l0pm (sub-macro to macroscopic) offers space for the movement of bone cells. Rising number of surgeries due to increasing incidence of orthopedic disorders, non-union fractures and injuries in the geriatric population is the prime driver for the bone grafts and substitutes market. In medical applications, orthopedic surgeons have been searching for porous, biocompatible structures with customizable geometry, high surface area ratio, and comparable mechanical strengths as a scaffold for bone grafts. Conventional manufacturing processes have been used for the manufacture of porous structures; however, they are incapable to control macrostructures. On the other hand, current additive manufacturing technology for ceramics can manipulate geometries, but rely on the gaps between printed material to create interconnected pores in 3D scaffolds. The gaps between printed lines usually leads to large pore size and poor integrity between layers. Reported herein is the 3D printing of hierarchical and porous hydroxyapatite (HAP) structures with interconnected pores from microscopic to macroscopic size created by combining freeze casting and extrusion-based 3D printing. This technique enables printing structures with no boundary between layers, therefore offering superior mechanical properties.

Described herein is a simultaneous extrusion and unidirectional freezing method to fabricate hierarchical and interconnected porous HAP structures for bone cell culture. In this methodology, pores are hierarchically integrated into one structure: macropores at the millimeter or submillimeter scale are controlled by the extrusion process, and micropores at the scale around lOpm are governed by freeze casting. The fabrication process is shown generally in Fig. 1, wherein (a) shows the printing setup, (b) shows the printing process, (c) shows the freeze casting process, (d) shows the final HA scaffold, and (e) shows the laminar pore and HAP structures. A syringe filled with HAP suspension is mounted on a customized printer and hence is able to move in a plane to print the predesigned patterns as is typical in additive manufacturing (Fig. 2, (a) and (b)). At the end of the syringe is a cone-shaped tip, where the suspension is extruded by applying air pressure. The extruded suspension is printed onto a cold plate and frozen from a highly viscous fluid into ice structures, as shown in Fig. 2, (c). After one layer of material is deposited, the surface is lowered for a new layer to be deposited upon the previous frozen layer to build up a 3D scaffold. During the freezing process, the ice crystals are nucleated from the bottom and travel along the temperature gradient. The HAP powders are squeezed together by the ice crystals, thus aligning along the temperature gradient (shown in Fig. 1, (c)). The ice structure is then placed in a -70 °C freezer for 12 hours to achieve thermal equilibrium. Hereafter the printed structure is sublimated by freeze drying, forming hierarchical structures with interconnected pores. Finally, the dried samples are sintered for the final products, as shown in Fig. 2, (d). This resulted in hierarchical patterns inside the printed filaments (Fig. 1, (d) and (e)) after freeze drying.

Central to the 3D printing of the hierarchical bone structure is the preparation of a suspension suitable for both extrusion and freeze casting. Extrusion requires the suspension to have a proper viscoelastic property so that the extruded material can keep its shape when it leaves the nozzle and is deposited. Freeze casting, on the other hand, requires the suspension to be flowable so that the hydroxyapatite particles inside can be squeezed during ice crystallization. The rheology behavior of the suspension was tested, as shown in Fig. 3. The suspension demonstrates the shear thinning pseudoplastic property. The shear stress, as shown in Fig. 4A, starts at around 100 Pa when the shear rate is zero, and then increases nonlinearly with shear rate. The stress t of a shear thinning fluid can be drawn as a function of shear rate g according to the Herschel-Bulkley model:

t = t_{n +}Kg

(1)

where t is the yield stress, K is consistency index, and n is flow index. The curve fitting resulted in parameters r,=78.84 Pa, K= 5.62 Pa-s" and «=0.64, with a determination coefficient of R²= 0.9997, indicating a good fit at room temperature. Correspondingly, the viscosity (Fig. 4B) decreases rapidly as the shear rate increases to 100 s ¹, followed by an approaching tendency to a low viscosity of 0.8 Pa-s. This shear thinning property allows the suspension behaving as a fluid at a high shear rate, typical 50-100 s ¹, when extruded through the nozzle, but acting as solid when it leaves the nozzle and is deposited onto the cold stage. From the perspective of energy, the loss modulus is higher than the storage modulus (Fig. 4C). Specifically, in the shear stress range between 78.84 Pa and 200 Pa, which is the typical fabrication conditions corresponding to shear rate between 0 to 100 s ¹, the storage modulus and loss modulus are around 40 Pa and 100 Pa, respectively, both decreasing with shear stress. The moduli of the suspension are much higher than those of the Newton fluids, which enable the extrusion process. However, the moduli of the suspension here are lower than the values in prior art materials (around 10³-10⁴). Thus, the prepared suspension is suitable for the proposed 3D printing of HAP structures described herein.

The final structures, with pores from macroscale to microscale after freeze drying to sublimate the ice and followed by sintering to enhance the bonding between particles, are presented in Fig. 5., wherein (a) shows 3D printed HA structure, (b) top surface morphology marked in (a), (c) top view of micropores in the marked region of (b). (d) and (e) show the top view of a single macropore (f) side view and (g) cross-section of the printed HAP structure (h) and (i) show the stable and unstable regions for the pore nucleation. The macropores are the holes with diameters ranging from submillimeter to millimeter level. They are designed and fabricated by the printing process and tailorable in a range depending on the suspension printability, freeze casting conditions, and printer parameters. The SEM images in Fig. 5, (b) and (c) show the micropore sizes from the top surface of the 3D printed HA structure from (a). More information of surface morphology is shown in Fig. 6. It has been found that patterns of micropores change with different freezing conditions. In the case where the initial temperature is 5°C and the decreasing rate is l°C min ¹, neighboring micropores are parallel to each other in the top view (Fig. 5, (b) and (e)). In another case where the initial temperature is -l8°C and the decreasing rate is l°C min ¹, neighboring micropores formed a snowflake pattern, as shown in Fig. 7. The freezing conditions greatly affect the patterns of the micropores.

During the freeze casting process, water freezes to ice and squeezes the HAP particles into contact with each other to form solid lamellae after sintering (Fig. 5, (i) and (h)). The cross- sectional structures are shown in Fig. 5, (f), (g), and (h). The volume ratio of the micropores is determined by the concentration of the suspension because the pores are exactly the replicates of the ice dendrites, while the shape and the distribution of the micropores are closely related to the temperature field. At the beginning of freeze casting, the suspension is firstly deposited on the cooling surface, the water phase is slowly cooled in all directions, resulting in randomly distributed and shaped pores showing the unstable region in Fig. 5, (i). The unstable crystallization is a transient process, with a height of only about 50pm. Hereafter the ice crystal reaches the stable zone, where the ice dendrites grow unidirectionally towards the temperature gradient. As a result, both the micropores and the HAP structures are in a lamella shape, thus forming interconnected pores (Fig. 5, (g) and (h)). More cross-sectional microstructure information can be seen in Fig. 8. The HAP lamella structures are shown in Fig. 9 by tilting the sample. The micropores are in a long canal shape, which is similar to the canals found in human cortical bones, demonstrating a width of ~ 1 Omih and along the whole cross-section of the printed porous structures. The interconnected micropores mimic human bone structures, therefore demonstrating the significance of the proposed printing technique. All of the hierarchical pores, ranging from lOpm to around lOmm, result in an increase of the surface area, hence improving the wettability upon exposure to liquid. In addition, the pores larger than the bone cell size would enable the migration of cells, therefore enhancing bone growth by providing higher surface area ratio for bone remodeling cells.

The mechanical properties of printed HAP structures are characterized by in-plane compression tests, as shown in Fig. 10 and Fig. 11. The compressive strength of the hierarchical porous structure is anisotropic, as shown in Fig. 10, (a) (showing stress-strain curves of the cylindrical sample and the extruded sample) with an ultimate strength of 22 and 2 MPa in parallel (middle line) and perpendicular (bottom line) to the freeze casting direction, respectively. This anisotropic property, which has also been observed in human bones, can be explained by the directional orientation of the pores. In comparison, the cylindrical sample shows an ultimate strength of 26 MPa in parallel to the freeze casting direction. This is easy to understand because macropores in the printed samples reduce the strength. For the cylindrical sample (the green line), the overall shape of the curve is similar to that of a typical ceramic, with a nearly linear increase when the strain increases, and a sharp drop once the ultimate strain is reached. On the other hand, the observed ultimate strain is 4.4%, much larger when compared to typical values of general ceramics of 0.1-0.2%. The printed hierarchical porous samples present significantly high strains at both directions when compared to freeze casted samples. The compressive strain goes up to 7.5% in the parallel direction and the strain reaches to 16.5% in the perpendicular direction. The deformation of the HAP laminar wall is schematically shown in Fig. 10, (b) and (c). As the load increases, cracks are initiated on the HAP laminar structures; the failed walls come into contact with each other due to the narrow pores, thus forming a more compact microstructure. Therefore, the printed samples are able to withstand additional load and reach ultrahigh ultimate strain. The printed HAP structures demonstrate high compression strength when compared to those from other 3D printing processes because of the high weight ratio of HAP, interconnected laminar structures, and superior structural integrity after sintering (as shown in Fig. 12).

The biocompatibility of the printed HAP scaffold with human cells and more importantly the cell migration in the pores was tested. The 3D scaffold samples were heated to 500 °C for 12 hours to remove volatile organics, autoclaved at l22°C and 16 psi for 15 minutes, and placed into individual CytoOne 6 well tissue culture plates with human mesenchymal stromal cells (MSC) and tissue culture medium per published MSC culture protocols. The cells and HAP scaffolds were placed in an incubator at 37 °C, 5% CO2, and 90% humidity for 3 to 5 days. The cultures plates were observed daily for interactions between the scaffolds and MSCs using phase contrast microscopy. At the end of the incubation period, the scaffolds are fixed using 4% freshly prepared paraformaldehyde in phosphate buffered saline and processed for observation using scanning electron microscopy. After cell growth, the microstructure on the top surface of the scaffold is shown in Fig. 13. The large-scale surface morphology is presented in (a) and (b). The cells on the top surface and in the pores are shown in (c) and (d), respectively. Widespread cell growth on the top surface can be seen in Fig. 14. (a) shows large scale surface morphology (b-f) show high density of cells on top surface (g-i) show single cell. The cells on the top surface demonstrate both cell adhesion and biocompatibility of the printed HAP scaffold.

The cell migration into the interconnected pores is shown in Fig. 15. The cross-section of laminated HAP wall structures was observed along two directions: vertical (Fig. 15, (a) and Fig. 16) and parallel (Fig 15, (b) and Figs. 17 and 18) to the laminar HAP wall structures. The inset picture in Fig. 15, (c) shows a cell grown on the top surface in (a). Fig. 15, (d) shows the middle portion of the cross-section, which is around 1.5 mm from the top surface. The cells in Fig. 15, (e) and (f) are from the marked region of Fig. 15, (d). The observed cells in both pictures demonstrate the success of cell migration along the pores, indicating these pores are interconnected. More details of cell growth and migration in the cross-section are provided by different locations in (g), (h), and (i) of the cross-section view along the parallel direction (j), (k), and (1) show cells growth in (g), (h), and (i), respectively. The bottom surface morphology of the printed HAP structure after cell growth is shown in Fig. 19. (a) and (b) show large scale surface morphology (c) shows cells growth on bottom surface (d) shows single cell growth in the marked region of (c). The success in cell growth shown in the cross section demonstrates the cell invasion and biocompatibility of the printed HAP scaffold. Specifically, the MSCs in the cross section prove that the pores are interconnected and their dimension permit cell invasion/migration. The multiscale pores result in an increase in the surface area and thus improved contact with ambient liquid in cell culture. In addition, the pores larger than the cell size will enhance cell growth by providing ample space. Conclusion

The results herein demonstrate the success of mimicking hierarchical human bone structure by integrating freeze casting and extrusion-based printing. This printing technique enables printing using a HAP suspension of high weight ratio, and eliminates the boundaries between layers, thus demonstrating high compressive strength for real applications. The printing also delivers interconnected HAP and porous laminar structures. The interconnected pores range from few micrometers to centimeter level, thus approaching the hierarchical pores in natural bones. This scaffold was biocompatible and permitted cell adhesion and migration. The interconnected micropores provides channels for the cells to grow into or invade the scaffold and greatly enhance the cell growth sites. The superior mechanical properties and cell culture performance demonstrate the great potential of the proposed 3D printing method for future bone tissue engineering.

Claims

CLAIMS:

1. A method of producing a porous hydroxyapatite structure, the method comprising: providing a pseudoplastic material comprising hydroxyapatite dispersed in an aqueous suspension;

depositing a first layer of the pseudoplastic material onto a surface;

cooling the first layer to a sufficient temperature so as to freeze water within the aqueous suspension of the first layer of the pseudoplastic material; and

depositing a second layer of the pseudoplastic material onto the first layer.

2. The method of claim 1, further comprising cooling the second layer to a sufficient temperature so as to freeze water within the aqueous suspension of the second layer of the pseudoplastic material.

3. The method of claim 2, further comprising depositing and freezing one or more additional layers of the pseudoplastic material onto the second layer.

4. The method of claim 2 or 3, further comprising freezing all layers of the pseudoplastic fluid material to form a solid intermediate structure comprising a solid ice phase and a solid hydroxyapatite phase.

5. The method of claim 4, further comprising freeze drying the solid intermediate structure, thereby removing the ice phase from the solid intermediate structure via sublimation and forming pores within the solid intermediate structure.

6. The method of claim 5, further comprising heating the solid intermediate structure to a temperature sufficient to sinter the hydroxyapatite phase.

7. The method of any one of claims 1 to 6, wherein the first layer of the pseudoplastic material is deposited onto the surface by extruding the pseudoplastic material through a nozzle.

8. The method of any one of claims 1 to 7, wherein the second layer of the pseudoplastic material is deposited onto the first layer by extruding the pseudoplastic material through a nozzle.

9. The method of any one of claims 1 to 8, wherein the pseudoplastic material comprises hydroxyapatite powder suspended in water.

10. The method of claim 9, wherein the pseudoplastic material comprises from about 40% to about 80% by weight of hydroxyapatite, based upon the total weight of the material taken as 100% by weight.

11. The method of any one of claims 1 to 10, wherein the pseudoplastic material comprises from about 0.1% to about 5% by weight of a dispersant, based upon the total weight of the material taken as 100% by weight.

12. The method of any one of claims 1 to 11, wherein the pseudoplastic material comprises one or more additional components selected from the group consisting of viscosifiers, defoamers, gelling agents, and pH adjusting agents.

13. The method of any one of claims 1 to 12, wherein the pseudoplastic material is an aqueous suspension having a pH of about 8 to about 10.

14. A porous hydroxyapatite structure formed by the method of any one of claims 1 to 13.

15. The porous hydroxyapatite structure of claim 14, wherein the structure comprises a plurality of interconnected macropores and micropores.

16. The porous hydroxyapatite structure of claim 15, wherein the plurality of macropores have a maximum cross-section of about 0.1 to about 5 mm, and/or wherein the plurality of micropores have a minimum cross-section of about 1 pm to about 50 pm.

17. The porous hydroxyapatite structure of any one of claims 14-16, wherein the structure has a compressive strength of at least about 10 MPa parallel to the plurality of channels.

18. A porous hydroxyapatite structure comprising a hydroxyapatite material and a plurality of interconnected macropores and micropores.

19. The porous hydroxyapatite structure of claim 18, wherein the plurality of macropores have a maximum cross-section of about 0.1 to about 5 mm, and/or wherein the plurality of micropores have a minimum cross-section of about 1 pm to about 50 pm.

20. The porous hydroxyapatite structure of claim 18 or 19, wherein the structure has a compressive strength of at least about 10 MPa parallel to the plurality of channels.

21. A method of growing cells in vitro comprising adding a quantity of cells and tissue culture medium to the porous hydroxyapatite structure of any one of claims 14 to 20 and culturing said cells under conditions suitable for cell growth.

22. A method of growing cells in vivo comprising introducing the porous hydroxyapatite structure of any one of claims 14 to 20 to into the body of a subject.