WO2010148106A1

WO2010148106A1 - Systems and methods involving calcium phosphate based materials

Info

Publication number: WO2010148106A1
Application number: PCT/US2010/038849
Authority: WO
Inventors: Susmita Bose; Ho Cheung Shum; Amit Bandyopadhyay; David A. Weitz
Original assignee: Washington State University Research Foundation; President And Fellows Of Harvard College
Priority date: 2009-06-16
Filing date: 2010-06-16
Publication date: 2010-12-23

Abstract

Provided are systems and methods for making materials (e.g., unique calcium phosphate based materials) using multiple emulsion microreactors. Certain aspects comprise surrounding a first fluid with a second fluid, surrounding the second fluid with a third fluid, wherein the first and third fluids are suitably immiscible with the second fluid, and wherein at least one of the fluids comprises a precursor(s) of the material, and forming, in at least one of the fluids, a desired material from the precursor(s). Certain aspects comprise surrounding a first fluid containing a calcium phosphate precursor with a second fluid, thus forming a droplet of the first fluid within the second fluid, and surrounding the second fluid with a third fluid, and wherein a unique calcium phosphate based material (e.g., having, portions with large aspect ratios, nanoscale porosity, relatively high BET surface area, relatively high pore volume, etc.) may be formed from the precursor(s).

Description

SYSTEMS AND METHODS INVOLVING CALCIUM PHOSPHATE BASED

MATERIALS

FIELD OF INVENTION Particular aspects related generally to material formation (e.g., calcium phosphate based materials), and in more particular aspects to systems and methods (e.g., micro fluidic techniques) for making calcium phosphate based materials, including, for example, forming of controlled porosity (e.g., nano-porous) calcium phosphate based material (e.g., mesoporous hydroxyapatite (HAp, (Caio, (PO4)6 (OH)₂) from calcium phosphate precursor material within multiple emulsion (e.g., double emulsion) microreactors (e.g., droplets).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to United States Provisional Patent Applications Serial No. 61/187,339, filed 16 June 2009 and entitled SYSTEMS AND METHODS INVOLVING CALCIUM PHOSPHATE BASED MATERIALS, incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH

This invention was made with government support under Grant Nos. DMR-060284 and CMMI-0728348 awarded by the National Science Foundation (NSF), Grant No. N00014-1-05- 0583 awarded by the Office of Naval Research and Grant No. R01-EB-007351 awarded by the National Institute of Health (NIH). The U.S. Government has certain rights in the invention.

BACKGROUND There is a pronounced need in the art for novel and effective methods to prepare materials from precursor(s) material to provide materials having unique properties and morphologies.

SUMMARY OF THE INVENTION Particular aspects provide systems and methods for making materials (e.g., unique calcium phosphate based materials) using multiple emulsion microreactors. Certain aspects comprise surrounding a first fluid with a second fluid, surrounding the second fluid with a third fluid, wherein the first and third fluids are suitably immiscible with the second fluid, and wherein at least one of the fluids comprises a precursor(s) of the material, and forming, in at least one of the fluids, a desired material from the precursor(s). Certain aspects comprise surrounding a first fluid containing a calcium phosphate precursor with a second fluid, thus forming a droplet of the first fluid within the second fluid, and surrounding the second fluid with a third fluid, and wherein a unique calcium phosphate based material (e.g., having, portions with large aspect ratios, nanoscale porosity, relatively high BET surface area, relatively high pore volume, etc.) may be formed from the precursor(s).

Systems and methods involving calcium phosphate based materials and their formation are generally described. The subject matter of the present invention involves, in some cases, interrelated products, alternative solutions to a particular problem, and/or a plurality of different uses of one or more systems and/or articles.

In some embodiments, a method of forming a calcium phosphate based material is described. The method may comprise, in some instances, surrounding a first fluid containing calcium phosphate precursor with a second fluid, and surrounding the second fluid with a third fluid. The method may further comprise, in some embodiments, forming a calcium phosphate based material from the calcium phosphate precursor.

In some embodiments, an article is described. The article may comprise, in some cases, a calcium phosphate based material, wherein the material comprises a portion with an aspect ratio of at least about 5:1, with a minor dimension defining the aspect ratio of less than about 1 micron. In some cases, the article may comprise a calcium phosphate based material, wherein the material comprises nanoscale pores. In some embodiments, at least about 10 vol% of the pores in the calcium phosphate based material have diameters of less than about 6 nm.

Particular aspects provide a method of forming a calcium phosphate based material, comprising: surrounding a first fluid with a second fluid; surrounding the second fluid with a third fluid, wherein the first and third fluids are suitably immiscible with the second fluid to provide a multiple emulsion microreactor, and wherein at least one of the first, second and third fluids comprise calcium phosphate precursor; and forming, in at least one fluid of the multiple emulsion microreactor, a calcium phosphate based material from the calcium phosphate precursor, wherein a method of forming a calcium phosphate based material is provided. Certain embodiments comprise providing to at least one of the first, second and third fluids of the multiple emulsion microreactor contains an additional reagent that facilitates formation of calcium phosphate from calcium phosphate precursor, and wherein the fluid to which the additional reagent is provided is different than the fluid containing the calcium phosphate precursor. In particular aspects, the additional reagent comprises alkaline material. In certain aspects, formation of calcium phosphate from calcium phosphate precursor comprises diffusion of the additional reagent into the fluid containing the calcium phosphate precursor. In certain embodiments, at least one of the first and third fluids contains the calcium phosphate precursor, and the additional reagent diffuses from the second fluid into the fluid containing the calcium phosphate precursor. In particular aspects, the second fluid contains the calcium phosphate precursor, and the additional reagent diffuses from at least one of the first and third fluids into the second fluid containing the calcium phosphate precursor. Certain aspects comprise a plurality of double emulsion microreactors, wherein the third fluid comprises a continuous phase contacting said plurality, and wherein the inner, first fluid contains calcium phosphate precursor. Particular embodiments comprise providing to at least one of the second and third fluids of the double emulsion microreactor an additional reagent that facilitates formation of calcium phosphate from calcium phosphate precursor, wherein formation of calcium phosphate from calcium phosphate precursor comprises diffusion of the additional reagent into the first fluid containing the calcium phosphate precursor. Certain embodiments provide a plurality of double emulsion microreactors, wherein the third fluid comprises a continuous phase contacting said plurality, and wherein the second fluid contains calcium phosphate precursor. Particular aspects comprise providing to at least one of the first and third fluids of the double emulsion microreactor an additional reagent that facilitates formation of calcium phosphate from calcium phosphate precursor, wherein formation of calcium phosphate from calcium phosphate precursor comprises diffusion of the additional reagent into the second fluid containing the calcium phosphate precursor.

In certain aspects, the osmolality or osmotic pressure within the fluid containing the calcium phosphate precursor is higher than the osmolality or osmotic pressure of at least one of the other fluids. Certain method embodiments comprise a plurality of double emulsion microreactors, wherein the third fluid comprises a continuous phase contacting said plurality, wherein the first fluid contains calcium phosphate precursor, the method comprising providing to the third continuous phase fluid an additional reagent that facilitates formation of calcium phosphate from calcium phosphate precursor, and wherein formation of calcium phosphate from calcium phosphate precursor comprises diffusion of the additional reagent into and through the second fluid and in turn into the fist fluid containing the calcium phosphate precursor. In particular aspects, the first and third fluids are aqueous based, the second fluid is oil based, and the osmolality or osmotic pressure within the first fluid containing the calcium phosphate precursor is higher than the osmolality or osmotic pressure of the third fluid. In particular aspects, the additional reagent comprises alkaline material.

In particular method aspects, at least one of the fluids contains, or receives an alkaline material. Certain methods aspects comprise, during formation of the calcium phosphate based material, increasing the volume occupied by the fluid containing the calcium phosphate precursor.

In preferred aspects, the multiple emulsion microreactor is a droplet multiple emulsion microreactor. In particular embodiments, the calcium phosphate based material comprises or consists essentially of at least one of hydroxyapatite and tricalcium phosphate, and β-tricalcium phosphate. In certain aspects, the morphology of the calcium phosphate based material comprises at least one of monodisperse, crystalline, mesoporous, nanoporous, needle-like nanoparticles, and microspheres comprising needle-like nanoparticles. In particular aspects, the calcium phosphate based material comprises at least one of: a portion with an aspect ratio of at least about 5 : 1 with a minor dimension defining the aspect ratio of less than about 1 micron; nanoscale pores; porosity wherein at least about 10 vol% of the pores have diameters of less than about 6 nm; porosity wherein at least about 20 vol% of the pores have diameters of less than about 6 nm; a BET surface area of at least about 150 m²/g; a total pore volume of at least about 0.25 mL/g; a total pore volume of at least about 0.50 mL/g; and a Ca to P ratio, by atomic number, of between about 1 :1 and about 2:1.

In certain aspects, the calcium phosphate material at least partially encapsulates or attaches to another material.

Additional aspects further comprise heat treating or calcining the calcium phosphate based material.

Further aspects provide a composition or article comprising a calcium phosphate based material prepared according to the described methods.

Yet additional aspects provide a composition or article of manufacture, comprising a calcium phosphate based material, wherein the calcium phosphate based material comprises at least one of: a portion with an aspect ratio of at least about 5:1 with a minor dimension defining the aspect ratio of less than about 1 micron; nanoscale pores; porosity wherein at least about 10 vol% of the pores have diameters of less than about 6 nm; porosity wherein at least about 20 vol% of the pores have diameters of less than about 6 nm; a BET surface area of at least about 150 m²/g; a total pore volume of at least about 0.25 mL/g; a total pore volume of at least about 0.50 mL/g; and a Ca to P ratio, by atomic number, of between about 1 : 1 and about 2:1. In particular aspects, the calcium phosphate based material comprises or consists essentially of at least one of hydroxyapatite and tricalcium phosphate, and β-tricalcium phosphate. In certain embodiments, the calcium phosphate material at least partially encapsulates or attaches to another material. In particular embodiments the compositions comprise heat-treated or calcined calcium phosphate based material.

Other advantages and novel features of the present invention will become apparent from the following detailed description of various non- limiting embodiments of the invention when considered in conjunction with the accompanying figures. In cases where the present specification and a document incorporated by reference include conflicting and/or inconsistent disclosure, the present specification shall control. If two or more documents incorporated by reference include conflicting and/or inconsistent disclosure with respect to each other, then the document having the later effective date shall control.

BRIEF DESCRIPTION OF THE DRAWINGS

Non- limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which in certain aspects are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention. In the figures:

FIG. 1 includes a schematic illustration of a system for producing calcium phosphate based material, according to one set of embodiments; FIG. 2 is an exemplary schematic illustration of a device used to produce multiple emulsions;

FIG. 3 is a schematic illustration of a device used to produce multiple emulsions, according to one set of embodiments;

FIG. 4 is an exemplary schematic illustration of the synthesis of calcium phosphate based material;

FIGS. 5A-5E include (A) an exemplary schematic illustration of a device for generating double emulsion droplets; and (B-E) exemplary optical microscope images of droplets containing calcium phosphate based materials; FIGS. E6-6C include optical microscope images of calcium phosphate based materials, according to one set of embodiments;

FIGS. 7A-7C include exemplary SEM images of calcium phosphate based materials;

FIGS. 8A-8B include TEM images of calcium phosphate based materials, according to one set of embodiments;

FIGS. 9A-9B include (A) exemplary adsorption/desorption isotherms and (B) exemplary pore size distributions for calcium phosphate based materials, according to one set of embodiments;

FIG. 10 includes thermogravimetric curves of calcium phosphate based materials, according to one set of embodiments; and

FIG. 11 includes a plot of exemplary x-ray diffraction patterns for calcium phosphate based materials.

DETAILED DESCRIPTION Systems and methods involving calcium phosphate based materials and their formation are generally described. In some embodiments, a method employing multiple emulsions is employed to form calcium phosphate based materials. The method may comprise surrounding a first fluid containing a calcium phosphate precursor with a second fluid, thus forming a droplet of the first fluid within the second fluid. The second fluid may be surrounded with a third fluid, and a calcium phosphate based material may be formed from the calcium phosphate precursor. In some embodiments, the calcium phosphate based material may have one or more desirable properties including, for example, portions with large aspect ratios, nanoscale porosity, relatively high BET surface area, and relatively high pore volume, among others.

The systems and methods described herein may be suitable for use in a variety of applications including, for example, orthopedic, dental, and other bio-related applications. As a specific example, calcium phosphate based materials such as hydroxyapatite may be used in medical implants. In addition, the systems and methods described herein may be used in catalyst applications. For example, calcium phosphate based materials are often used in conjunction with different biomolecules to provide surfaces for adsorption and catalysis (e.g., for biochemical reactions).

As used herein, a "calcium phosphate based material" generally refers to materials comprising at least about 30% calcium or phosphate material by mass. In some embodiments, a calcium phosphate based material may comprise at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% of calcium and phosphate by mass, or more. Calcium phosphate based materials include a variety of forms including, for example, hydroxyapatite ( HAp, (Ca)₁₀ (PO₄)S(OH)₂ ); tricalcium phosphate ( TCP, Ca3(POzt)₂ ) including alpha, beta, and gamma TCP; calcium dihydrogen phosphate ( Ca(H₂PO₄)₂ ); calcium hydrogen phosphate ( CaHPO₄ ); tetracalcium phosphate ( Ca₄O(PO₄)₂ ); amorphous calcium phosphate ( Cai₀-_xH_2x(PO₄)6(OH)₂ ); octacalcium phosphate ( CagH₂(PO₄)6 ^• 5H₂O ); dicalcium phosphate dihydrate ( CaHPO₄ ^• 2H₂O ); dicalcium phosphate ( CaHPO₄ ); calcium pyrophosphate ( Ca₂P₂O₇ ) including alpha, beta, and gamma; calcium pyrophosphate dehydrate ( Ca₂P₂O₇ ^• 2H₂O ); heptacalcium phosphate ( Ca₇(P₅Oi₆)₂ ); tetracalcium dihydrogen phosphate ( Ca₄H₂Pδ0₂o ); monocalcium phosphate monohydrate ( Ca(H₂PO₄)₂ ^• H₂O ); calcium metaphosphate ( Ca(POs)₂ ) including alpha, beta, and gamma; and the like. In some embodiments, the calcium phosphate based material may have a Ca to P ratio, by atomic number, of between about 1 : 1 and about 2: 1. The calcium phosphate based material may be amorphous or crystalline. In some embodiments, at least a portion of the calcium phosphate based material is crystalline. In some cases, substantially all (e.g., at least about 80 wt%, at least about 90 wt%, at least about 95 wt%, at least about 98 wt%, at least about 99 wt%, or more) of the calcium phosphate based material is crystalline.

As used herein, "calcination" refers to a process in which a material is heated to a temperature below its melting point to effect a thermal decomposition or a phase transition other than melting. In one aspect, a method of forming a calcium phosphate based material is described.

FIG. IA includes a schematic illustration of system 10 which may be used to form calcium phosphate based materials. In this exemplary embodiment, first fluid 12, which contains calcium phosphate precursor, is surrounded with second fluid 14, to form a droplet containing calcium phosphate precursor. Second fluid 14 may be immiscible with first fluid 12. Second fluid 14 may be surrounded with third fluid 16. Third fluid 16 may be immiscible with second fluid 14. The first, second, and third fluids shown in FIGS. 1A-1B may form a double emulsion, described in more detail below. As shown in FIG. IB, a calcium phosphate material 20 may be formed from the calcium phosphate precursor in first fluid 12, for example, by the addition of an alkaline material to the system. The use of a multiple emulsion based system, as shown in the exemplary double emulsion embodiment of FIGS. 1A-1B, allows for the production of calcium phosphate based materials with unique properties (e.g., porosity, aspect ratio, BET surface area, density, structure, etc.), described in more detail below. In addition, the use of multiple emulsions allows for precise control of parameters such as the concentration of a species within a fluid, rate of addition of a species to a fluid, volume of a fluid phase (e.g., volume of a droplet in which a calcium phosphate material is formed), and the like. In some embodiments, the use of multiple emulsions allows for the isolation of one or more inner fluids from other inner fluids, thus preventing unwanted agglomeration of calcium phosphate based materials. For example, a middle fluid (e.g., second fluid 14 in FIGS. 1A-1B) may completely surround an inner fluid (e.g., first fluid 12 in FIGS. 1A-1B) such that the inner fluid is isolated from other inner fluids 12 (e.g., droplets) which may contain calcium phosphate precursor. In such cases, the calcium phosphate based material formed in the first inner fluid may not contact the calcium phosphate based material formed in another inner fluid, preventing agglomeration thereof. With respect to calcium phosphate embodiments, a variety of calcium and/or phosphate precursors may be used in association with the exemplary systems and methods described herein. For example, calcium salts (e.g., calcium nitrate tetrahydrate (Ca(NOs)₂^H₂O)) may be used, in some cases, as a source of calcium. Sources of phosphate may comprise, for example, phosphoric acid, phosphate salts, and the like. In some embodiments, at least one of the fluids may contain an alkaline material. The alkaline material may facilitate and/or be used to form the calcium phosphate based material from the calcium phosphate precursor. For example, in some embodiments, forming a calcium phosphate based material comprises adding an alkaline material to at least one of the fluids. For example, with respect to Figs. IA and IB, the alkaline material may be added to the third fluid 16 (e.g., the continuous phase), from which it may diffuse (e.g., through the second fluid 14) into the first fluid 12. This diffusion may occur, for example, if the osmolality or osmotic pressure within the first fluid is higher than the osmolality or osmotic pressure in the third fluid. In some cases, the infusion of material and/or water (in the case of aqueous based third fluids) from the third fluid to the first fluid may result in an increase in the volume occupied by the first fluid receiving said infusion.

Once formed, the calcium phosphate based material may be further processed to produce desirable characteristics. In some embodiments, the calcium phosphate material may be calcined (e.g., at above about 200 ⁰C, at above about 300 ⁰C, at above about 400 ⁰C, at above about 500 ⁰C, at above about 600 ⁰C, at above about 700 ⁰C, at above about 800 ⁰C, at above about 900 ⁰C, or higher), for example, to produce a crystalline calcium phosphate based material from an at least partially amorphous calcium phosphate based material.

In some embodiments, the calcium phosphate based material may serve as a support for a catalyst or other agent or moiety deposited on and/or within the material. In some embodiments, at least a portion of the calcium phosphate based material has an aspect ratio of at least about 5 : 1 , at least about 10 : 1 , at least about 25 : 1 , or greater. In some cases, at least a portion of the calcium phosphate based material has an aspect ratio of between about 1 : 1 and about 5: 1, and/or at least a portion of the calcium phosphate based material has an aspect ratio of between between about 5 : 1 and about 10: 1. As used herein, the "aspect ratio" of a material (or a portion of a material) may be calculated as the ratio of the longest trajectory to the shortest trajectory through the material, from one boundary to another. It should be noted that the longest and shortest trajectories may be, but are not necessarily, perpendicular to each other. In some embodiments, a calcium phosphate based material may have any of the aspect ratios mentioned above while having a shortest trajectory through the material of less than about 1 micron, less than about 500 nm, less than about 250 nm, less than about 100 nm, less than about 50 nm, less than about 10 nm, less than about 1 nm, or smaller. In some cases, a calcium phosphate based material may have any of the aspect ratios mentioned above while having a shortest trajectory through the material of between about 1 nm and about 1 micron, between about 10 nm and about 1 micron, between about 100 nm and about 1 micron, between about 1 nm and about 500 nm, between about 10 nm and about 500 nm, between about 100 nm and about 500 nm, between about 1 nm and about 250 nm, between about 10 nm and about 250 nm, or between about 100 nm and 250 nm, between about 1 nm and about 100 nm, or between about 10 nm and about 100 nm. In some embodiments, the calcium phosphate based material may comprise relatively small particles (e.g., crystals, amorphous particles). In addition, one or more droplets used in the methods described herein may be relatively small in some embodiments. For example, exemplary calcium phosphate based materials and/or droplets described herein may have a maximum cross-sectional diameters of less than about 100 microns, less than about 10 microns, less than about 1 micron, less than about 100 nm, less than about 10 nm, less than about 1 nm, or smaller. In some embodiments, a plurality of calcium phosphate based materials and/or droplets described herein may have a maximum cross-sectional diameters of less than about 100 microns, less than about 10 microns, less than about 1 micron, less than about 100 nm, less than about 10 nm, less than about 1 nm, or smaller. As used herein, the "maximum cross-sectional dimension" refers to the largest distance between two opposed boundaries of an individual structure (e.g., calcium phosphate based material or droplet) that may be measured. The "average maximum cross-sectional dimension" of a plurality of structures refers to the number average. In some embodiments, the calcium phosphate based materials and/or the emulsions containing calcium phosphate based materials, and/or calcium phosphate precursors, may be substantially the same shape and/or size ("monodisperse"). For example, the calcium phosphate based materials and/or emulsions may have a distribution of dimensions such that no more than about 10% of the calcium phosphate based materials and/or emulsions have a maximum cross- sectional dimension that varies by more than about 10% of the average maximum cross- sectional dimension of the calcium phosphate based materials and/or emulsions, and in some cases, such that no more than about 8%, about 5%, about 3%, about 1%, about 0.3%, about 0.1%, about 0.03%, or about 0.01% have a maximum cross-sectional dimension that varies by more than about 10% of the average maximum cross-sectional dimension of the calcium phosphate based materials and/or emulsions. Those of ordinary skill in the art will be able to determine the average maximum cross-sectional dimension of a plurality or series of droplets, emulsions, or particles, for example, using laser light scattering, microscopic examination, or other known techniques. The calcium phosphate based material may be porous, substantially porous, substantially non-porous, and/or non-porous. The pores may comprise a range of sizes and/or be substantially uniform in size. In some cases, the pores may or might not be visible using imaging techniques (e.g., scanning electron microscope). The pores may be open and/or closed pores.

In some embodiments, the calcium phosphate based material may comprise nanoscale pores. In some cases, at least about 5 vol%, at least about 10 vol%, at least about 20 vol%, or more of the pores in the calcium phosphate based material have diameters of less than about 6 nm. In some cases, at least about 5 vol%, at least about 10 vol%, at least about 20 vol%, at least about 30 vol%, at least about 35 vol% or more of the pores in the calcium phosphate based material have diameters of less than about 10 nm. In some cases, at least about 10 vol%, at least about 20 vol%, at least about 30 vol%, at least about 40 vol%, at least about 50 vol%, at least about 60 vol% or more of the pores in the calcium phosphate based material have diameters of less than about 20 nm. In some cases, at least about 10 vol%, at least about 25 vol%, at least about 50 vol%, at least about 75 vol%, at least about 85 vol%, at least about 90 vol% or more of the pores in the calcium phosphate based material have diameters of less than about 80 nm. In some embodiments, at least about 10 vol%, at least about 25 vol%, at least about 50 vol%, at least about 75 vol%, at least about 85 vol%, at least about 90 vol%, at least about 95 vol%, at least about 98 vol% or more of the pores in the calcium phosphate based material have diameters of less than about 120 nm. The number of pores with a pore size less than a given dimension may be calculated on a volume percentage by dividing the total volume occupied by the pores with a size under the given dimension by the total amount of volume occupied by all of the pores. One of ordinary skill in the art will be able to calculate pore-size and pore-volume distributions using, for example, nitrogen adsorption and the Barrett- Joyner-Halenda (BJH) method of isotherm analysis. In addition, BET surface area analysis may also be used. Suitable methods for making such measurements are described, for example, in "Adsorption isotherms of microporous-mesoporous solids revisited" in Applied Catalysis A: General, Vol. 129, Iss. 2, August 31, 1995, Pages 157-165, which is incorporated herein by reference in its entirety.

The calcium phosphate based materials described herein may have a relatively high pore volume, in some embodiments. For example, in some cases, the calcium phosphate based material has a total pore volume of at least about 0.01 mL/g, at least about 0.1 mL/g, at least about 0.25 mL/g, at least about 0.5 mL/g, or more.

In some embodiments, the calcium phosphate based materials may have a high BET surface area. In some cases, the BET surface area of the calcium phosphate based materials may be greater than about 1 m²/g, greater than about 5 m²/g, greater than about 10 m²/g, greater than about 20 m²/g, greater than about 30 m²/g, greater than about 50 m²/g, greater than about 100 m²/g, greater than about 150 m²/g, greater than about 200 m²/g, or greater. BET surface area measurements are known to those of ordinary skill in the art, and are described generally in Principles of Ceramics Processing by James Reed, ISBN: 978-0-471-59721-6, and S. Brunauer, P. H. Emmett, and E. Teller, J. Am. Chem. Soc, 1938, 60, 309, which are incorporated herein by reference in their entirety.

The porosity of a calcium phosphate based material may be measured as a percentage or fraction of the void spaces in the calcium phosphate based material. The percent porosity of a calcium phosphate based material may be measured using techniques commonly known to those of ordinary skill in the art, for example, using volume/density methods, water saturation methods, water evaporation methods, mercury intrusion porosimetry methods, and nitrogen gas adsorption methods. In some embodiments, the calcium phosphate based material may be at least about 10% porous, at least about 20% porous, at least about 30% porous, at least about 40% porous, at least about 50% porous, at least about 60% porous, or greater. The pores may be open pores (e.g., have at least one part of the pore is open an outer surface of the calcium phosphate based material and/or another pore) and/or closed pores (e.g., the pore does not comprise an opening to an outer surface of the calcium phosphate based material or another pore). In some cases, the pores of a calcium phosphate based material may consist essentially of open pores (e.g., the pores of the calcium phosphate based material are greater than at least 70%, greater than at least 80%, greater than at least 90%, greater than at least 95%, or greater, open pores). In some cases, only a portion of the calcium phosphate based material may be substantially porous. For example, in some cases, only a single surface of the calcium phosphate based material may be substantially porous. As another example, in some cases, the outer surface of the calcium phosphate based material may be substantially porous and the inner core of the calcium phosphate based material may be substantially non-porous. In a particular embodiment, the entire calcium phosphate based material is substantially porous.

In some embodiments, the calcium phosphate based materials may be formed such that they are deposited on another material. For example, a material (e.g., a metal, organic material, etc.) may be contained within an inner fluid of a multiple emulsion. In addition, a calcium phosphate precursor may also be contained within the inner fluid. The calcium phosphate precursor may then be deposited on the material to form a coating of calcium phosphate based material, for example, via the addition of an alkaline material.

In some embodiments, the material on which the calcium phosphate based material is deposited may comprise another calcium phosphate based material (which may be the same as or different than the calcium phosphate based material that is deposited). Calcium phosphate based materials may also be deposited on non-calcium phosphate based materials. Calcium phosphate based materials may, in some cases, partially encapsulate a material. In other instances, calcium phosphate based materials may substantially fully encapsulate a material. For example, particles can be suspended in the first fluid containing calcium phosphate precursor, and the precursor can be deposited on the particles (e.g., upon adding an alkaline substance). Examples of non-calcium phosphate based materials on which calcium phosphate based materials could be deposited include, for example, organic materials (e.g., polymers), metals (e.g., metal nanoparticles), semiconductors (e.g., quantum dots), other inorganic materials, and the like. In some embodiments, the calcium phosphate based material may be at least partially calcined after being deposited on a material.

Those of ordinary skill in the art will be able, based on the teachings of this disclosure, to select appropriate conditions for making the materials described herein that will result in the production of materials possessing one or more of any of the properties described herein, as well as other parameters and properties not described herein. For example, one of ordinary skill in the art will be capable of selecting a concentration of alkaline material such that a desired porosity is produced in the calcium phosphate based material. As another example, one of ordinary skill in the art can select an appropriate droplet size to produce a desired particle size. Varying (e.g., increasing or decreasing) the concentrations of calcium and/or phosphorous precursors in the inner fluid can, for example, be used to tune at least one of the structure, density, and porosity characteristics of the calcium phosphate based materials. It should also be understood that any of the properties of the calcium phosphate based materials may be present, alone or in combination with each other, in a single material.

In addition, although systems and methods for the production of calcium phosphate based materials have been described, it should be understood that the embodiments described herein may be applied more broadly and with much greater control, to a wide variety of inorganic materials. One of ordinary skill in the art can determine appropriate system parameters (e.g., temperature(s), concentration(s), droplet size, etc.) that could be used to produce inorganic materials with favorable and/or desired properties using the methods described herein.

Some embodiments involve the use of multiple emulsions. A "multiple emulsion," as used herein, describes or comprises larger droplets that contain one or more smaller droplets therein. The larger droplet or droplets may be suspended in a third fluid in some cases. In certain embodiments, emulsion degrees of nesting within the multiple emulsion are possible. For example, an emulsion may contain droplets containing smaller droplets therein, where at least some of the smaller droplets contain even smaller droplets therein, etc. In some embodiments, the inner fluid of a multiple emulsion may act as an enclosed volume in which calcium phosphate based materials may be formed, as described, for example in FIGS. IA- IB. In some cases, the multiple emulsions described herein may be made in a single step using different fluids. In one set of embodiments, a triple emulsion may be produced, i.e., an emulsion containing a first fluid (e.g., a fluid containing calcium phosphate precursor), surrounded by a second fluid, which in turn is surrounded by a third fluid. In some cases, the third fluid and the first fluid may be the same. These fluids can be referred to as an inner fluid (IF), a middle fluid (MF) and an outer fluid (OF), respectively, and are often of varying miscibilities due to differences in hydrophobicity. For example, the inner fluid may be water soluble, the middle fluid oil soluble, and the outer fluid water soluble. This arrangement is often referred to as a w/o/w multiple emulsion ("water/oil/water"). Another multiple emulsion may include an inner fluid that is oil soluble, a middle fluid that is water soluble, and an outer fluid that is oil soluble. This type of multiple emulsion is often referred to as an o/w/o multiple emulsion ("oil/water/oil"). It should be noted that the term "oil" in the above terminology merely refers to a fluid that is generally more hydrophobic and not miscible in water, as is known in the art. Thus, the oil may be a hydrocarbon in some embodiments, but in other embodiments, the oil may comprise other hydrophobic fluids. As used herein, two fluids are immiscible, or not miscible, with each other when one is not soluble in the other to a level of at least 10% by weight at the temperature and under the conditions at which the two fluids are mixed in producing the calcium phosphate based materials (e.g., under the conditions at which a multiple emulsion is produced). For instance, the fluid and the liquid may be selected to be immiscible within the time frame of the formation of the fluidic droplets. In some embodiments, the inner and outer fluids are compatible, or miscible, while the middle fluid is incompatible or immiscible with each of the inner and outer fluids. In other embodiments, however, all three fluids may be mutually immiscible, and in certain cases, all of the fluids do not all necessarily have to be water soluble. In still other embodiments, additional fourth, fifth, sixth, etc., fluids may be added to produce increasingly complex droplets within droplets, e.g., a first fluid may be surrounded by a second fluid, which may in turn be surrounded by a third fluid, which in turn may be surrounded by a fourth fluid, etc.

In the descriptions herein, multiple emulsions are generally described with reference to a three phase system, i.e., having an outer fluid, a middle fluid, and an inner fluid (which may contain, for example, a calcium phosphate precursor material). However, it should be noted that this is by way of example only, and that in other systems, additional fluids may be present within the multiple droplet. As examples, an emulsion may contain a first fluid droplet and a second fluid droplet, each surrounded by a third fluid, which is in turn surrounded by a fourth fluid; or an emulsion may contain multiple emulsions with higher degrees of nesting. Accordingly, it should be understood that the descriptions of the inner fluid, middle fluid, and outer fluid are by ways of ease of presentation, and that the descriptions below are readily extendable to systems involving additional fluids. In addition, calcium phosphate precursor may be contained in any one of the fluids, which may be used, for example, to form calcium phosphate based materials in any of the fluids. For example, the middle fluid may contain calcium phosphate precursor, in some cases, which may be used to form a shell of calcium phosphate based material.

In one set of embodiments, multiple emulsions are formed by flowing three (or more) fluids through a system of conduits. The system may be a microfluidic system. "Microfluidic," as used herein, refers to a device, apparatus or system including at least one fluid channel having a cross-sectional dimension of less than about 1 millimeter (mm), and in some cases, a ratio of length to largest cross-sectional dimension of at least 3: 1. One or more conduits of the system may be a capillary tube. In some cases, multiple conduits are provided, and in some embodiments, at least some are nested, as described herein. The conduits may be in the microfluidic size range and may have, for example, average inner diameters, or portions having an inner diameter, of less than about 1 millimeter, less than about 300 micrometers, less than about 100 micrometers, less than about 30 micrometers, less than about 10 micrometers, less than about 3 micrometers, or less than about 1 micrometer, thereby providing droplets having comparable average diameters. One or more of the conduits may (but not necessarily), in cross section, have a height that is substantially the same as a width at the same point. Conduits may include an orifice that may be smaller, larger, or the same size as the average diameter of the conduit. For example, conduit orifices may have diameters of less than about 1 mm, less than about 500 micrometers, less than about 300 micrometers, less than about 200 micrometers, less than about 100 micrometers, less than about 50 micrometers, less than about 30 micrometers, less than about 20 micrometers, less than about 10 micrometers, less than about 3 micrometers, etc. In cross-section, the conduits may be rectangular or substantially non-rectangular, such as circular or elliptical. The conduits of the present invention can also be disposed in or nested in another conduit, and multiple nestings are possible in some cases. In some embodiments, one conduit can be concentrically retained in another conduit and the two conduits are considered to be concentric. In other embodiments, however, one conduit may be off-center with respect to another, surrounding conduit. By using a concentric or nesting geometry, the inner and outer fluids, which are typically miscible, may avoid contact facilitating great flexibility in making multiple emulsions and in practicing and optimizing techniques for encapsulation and polymerosome formation. For example, this technique allows for fabrication of core-shell structure, and these core-shell structures can be converted into capsules. In some cases, the capsules may contain calcium phosphate based materials, either within the interior volume of the capsule, within the shell of the capsule, or within both.

As the systems described herein may be truly three-dimensional microfluidic devices, e.g., having concentric conduit arrangements, the inner fluid can be completely shielded from the outer fluid in certain embodiments. This may reduce or eliminate problems that can occur in other systems, when the inner and outer fluid may contact each other at or near a solid surface, such as in a two-dimensional system. This also may allow for the controlled addition of a material from an outer fluid to an inner fluid. For example, an alkaline material may be dissolved in an outer fluid and added to an inner fluid at a controlled rate. A flow pathway can exist in an inner conduit and a second flow pathway can be formed in a coaxial space between the external wall of the interior conduit and the internal wall of the exterior conduit, as discussed in detail below. The two conduits may be of different cross- sectional shapes in some cases. In one embodiment, a portion or portions of an interior conduit may be in contact with a portion or portions of an exterior conduit, while still maintaining a flow pathway in the coaxial space. Different conduits used within the same device may be made of similar or different materials. For example, all of the conduits within a specific device may be glass capillaries, or all of the conduits within a device may be formed of a polymer, for example, polydimethylsiloxane, as discussed below. A geometry that provides coaxial flow can also provide hydrodynamic focusing of that flow, according to certain embodiments of the invention. Many parameters of the droplets, both inner droplets and middle layer droplets (outer droplets) can be controlled using hydrodynamic focusing. For instance, droplet diameter, outer droplet thickness and the total number of inner droplets per outer droplet can be controlled. Multiple emulsion parameters can also be engineered and/or tuned by adjusting, for example, the system geometry, the flowrate of the inner fluid, the flowrate of the middle fluid and/or the flowrate of the outer fluid. By controlling these three flow rates independently, the number of internal droplets and the shell thickness of the outer droplet (middle fluid) can be predicatively chosen and/or optimized. FIG. 2 includes an exemplary schematic illustration of a device 100 for producing multiple emulsions. Device 100 has an outer conduit 110, a first inner conduit (or injection tube) 120, and a second inner conduit (or collection tube) 130. An inner fluid 140 (which may contain, for example, a calcium phosphate precursor) is shown flowing in a right to left direction and middle fluid 150 flows in a right to left direction in the space outside of injection tube 120 and within conduit 110. Outer fluid 160 flows in a left to right direction in the pathway provided between outer conduit 110 and collection tube 130. After outer fluid 160 contacts middle fluid 150, it changes direction and starts to flow in substantially the same direction as the inner fluid 140 and the middle fluid 150, right to left. Injection tube 120 includes an exit orifice 164 at the end of tapered portion 170. Collection tube 130 includes an entrance orifice 162, an internally tapered surface 172, and exit channel 168. Thus, the inner diameter of injection tube 120 decreases in a direction from right to left, as shown, and the inner diameter of collection tube 130 increases from the entrance orifice in a direction from right to left. These constrictions, or tapers, can provide geometries that aid in producing consistent multiple emulsions. The rate of constriction may be linear or non-linear. As illustrated in FIG. 2, inner fluid 140 exiting from orifice 164 can be completely surrounded by middle fluid 150, as there is no portion of inner fluid 140 that contacts the inner surface of conduit 110 after its exit from injection tube 120. Thus, for a portion between exit orifice 164 to a point inside of collection tube 130 (to the left of entrance orifice 162), a stream of fluid 140 is concentrically surrounded by a stream of fluid 150. Additionally, middle fluid 150 may not come into contact with the surface of collection tube 130, at least until after the multiple emulsion has been formed, because it is concentrically surrounded by outer fluid 160 as it enters collection tube 130. Thus, from a point to the left of exit orifice 164 to a point inside of collection tube 130, a composite stream of three fluid streams is formed, including inner fluid 140 concentrically surrounded by a stream of middle fluid 150, which in turn is concentrically surrounded by a stream of outer fluid 160. The inner and middle fluids do not typically break into droplets until they are inside of collection tube 130 (to the left of entrance orifice 162). Under "dripping" conditions, the droplets are formed closer to the orifice, while under "jetting" conditions, the droplets are formed further downstream, i.e., to the left as shown in FIG. 2. In some cases, such as when droplets of middle fluid 150 (outer droplets) are formed at the same rate as are droplets of inner fluid 140, then there is a one-to-one correspondence between inner fluid and middle fluid droplets, and each droplet of inner fluid is surrounded by a droplet of middle fluid, and each droplet of middle fluid contains a single inner droplet of inner fluid, as illustrated in FIGS. 1A-1B. Such a configuration may be useful, for example, in observing the formation of a calcium phosphate based material within an inner fluid. In addition, in some embodiments, such a configuration may prevent unwanted agglomeration of calcium phosphate based materials. The term "outer droplet," as used herein, typically means a fluid droplet containing an inner fluid droplet that comprises a different fluid. In many embodiments that use three fluids for multiple emulsion production, the outer droplet is formed from a middle fluid and not from the outer fluid as the term may imply. It should be noted that the above-described figure is by way of example only, and other devices are also contemplated within the instant invention. For example, the device in FIG. 2 may be modified to include additional concentric tubes, for example, to produce more highly nested droplets. For instance, in FIG. 3, a device having three concentric tubes is shown, which may be used to produce nested fluidic droplets having an inner fluid, a first middle fluid surrounding the inner fluid, and a second inner fluid surrounding the first middle fluid. Even higher degrees of nesting are possible, for example, 4 concentric tubes, 5 concentric tubes, or the like. It should be noted that "concentric," as used herein, does not necessarily refer to tubes that strictly coaxial, but also includes nested or "off-center" tubes that do not share a common center line. Droplet formation and morphology can be affected in a number of ways. For example, the geometry of the device, including the relationship of an outer conduit and two inner conduits, can be useful in developing multiple emulsions of desired size, frequency, and content. For example, the volume of the inner fluid may be controlled, and thus, in some cases, the size of the calcium phosphate based material that is formed may be controlled. For example, the size of the orifice 162 and the inner taper of collection tube 130 can help to maintain three fluids in position, allowing droplets 180 to form. In addition, droplet formation can be affected by the rate of flow of the inner fluid, the rate of flow of the middle fluid, the rate of flow of the outer fluid, the total amount of flow or a change in the ratios, and/or combinations of any of these flow rates. In some embodiments, multiple droplets of inner fluid can be formed within a single droplet of the middle fluid. For example, 2, 3, 4, 5, 10, 30, 100, 300, 1000 or more droplets of inner fluid can be formed within a droplet of middle fluid by varying the frequency of droplet formation of either (or both) the inner fluid or the middle fluid, in relation to the other of the inner fluid or the middle fluid. For example, if the velocity of the inner fluid is altered so that five droplets are formed over the same amount of time as a single droplet of middle fluid, then a droplet of middle fluid may contain, on average, five droplets of inner fluid. It should be noted that, depending on the fluid flow characteristics, some of the middle fluid droplets may contain more or fewer droplets of inner fluid, although the average is five droplets, as discussed in this example. As the absolute and relative flow rates of the three fluids can be carefully controlled using the devices described herein, the middle fluid droplets containing specific numbers of inner fluid droplets can be consistently and repeatedly formed. In some embodiments, the standard deviation from a target number of inner fluid droplets per middle fluid droplet may be, for example, less than one inner droplet, or less than 20% of the number of inner droplets per middle fluid droplet. In other embodiments, the standard deviation may be, for example, less than 15%, less than 12%, less than 10%, less than 8%, or less than 6% of the number of inner droplets per middle fluid droplet.

The relative sizes of the inner fluid droplet and the middle fluid droplet can also be carefully controlled, i.e., the ratio of the size of the inner and outer droplets can be predicatively controlled. By controlling this ratio, the rate at which a material (e.g., an alkaline material) diffuses into the inner fluid may be controlled, in some cases. For instance, inner fluid droplets may fill much of or only a small portion of the middle fluid (outer) droplet. Inner fluid droplets may fill less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 30%, less than 20%, or less than 10% of the volume of the outer droplet. Alternatively, the inner fluid droplet may form greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 90%, 95%, or 99% of the volume of the outer droplet. In some cases, the outer droplet can be considered a fluid shell, or coating, when it contains an inner droplet, as some or most of the outer droplet volume may be filled by the inner droplet. The ratio of the middle fluid shell thickness to the middle fluid droplet radius can be equal to or less than, e.g., 5%, 4%, 3%, or 2%. This can allow, in some embodiments, for the formation of multiple emulsions with only a very thin layer of material separating, and thus stabilizing, two miscible fluids. The middle shell material can also be thickened to greater than or equal to, e.g., 10%, 20%, 30%, 40%, or 50% of the middle fluid droplet radius.

The rate of production of multiple emulsion droplets may be determined by the droplet formation frequency, which under many conditions can vary between approximately 100 Hz and 5000 Hz. In some cases, the rate of droplet production may be at least about 200 Hz, at least about 300 Hz, at least about 500 Hz, at least about 750 Hz, at least about 1,000 Hz, at least about 2,000 Hz, at least about 3,000 Hz, at least about 4,000 Hz, or at least about 5,000 Hz.

Production of large quantities of multiple emulsion products, e.g. calcium phosphate based materials within emulsions, can be facilitated by the parallel use of multiple devices in some instances. In some cases, relatively large numbers of devices may be used in parallel, for example at least about 10 devices, at least about 30 devices, at least about 50 devices, at least about 75 devices, at least about 100 devices, at least about 200 devices, at least about 300 devices, at least about 500 devices, at least about 750 devices, or at least about 1,000 devices or more may be operated in parallel. The devices may comprise different conduits (e.g., concentric conduits), orifices, micro fluidics, etc. In some cases, an array of such devices may be formed by stacking the devices horizontally and/or vertically. The devices may be commonly controlled, or separately controlled, and can be provided with common or separate sources of inner, middle, and outer fluids, depending on the application. In some embodiments of the invention, a hardened shell may be formed around an inner droplet, such as by using a middle fluid that can be solidified or gelled. In this way, capsules can be formed with consistently and repeatedly-sized inner droplets, as well as a consistent and repeatedly-sized outer shell. In some embodiments, this can be accomplished by a phase change in the middle fluid. A "phase change" fluid is a fluid that can change phases, e.g., from a liquid to a solid. A phase change can be initiated by a temperature change, for instance, and in some cases the phase change is reversible. For example, a wax or gel may be used as a middle fluid at a temperature which maintains the wax or gel as a fluid. Upon cooling, the wax or gel can form a solid or semisolid shell, e.g., resulting in a capsule. The shell may also be a bilayer, such as can be formed from two layers of surfactant. In another embodiment, the shell can be formed by polymerizing the middle fluid droplet. This can be accomplished in a number of ways, including using a pre -polymer that can be catalyzed, for example, chemically, through heat, or via electromagnetic radiation (e.g., ultraviolet radiation) to form a solid polymer shell. In another set of embodiments, fluid can be removed from an inner droplet in order to, for example, concentrate any species that may be contained within the inner droplet. Fluid may be removed from the inner droplet, or the inner droplet may be concentrated, using techniques similar to those described herein for removing fluid from an outer droplet. For instance, fluid can diffuse from or evaporate out of the inner droplet in order to reduce the size of the inner droplet, and therefore concentrate any components of the inner droplet that do not substantially diffuse or evaporate. For example, the volume of an inner droplet can be reduced by more than 50%, 75%, 90%, 95%, 99%, or 99.9%. Thus, the core radius of the inner droplet can be reduced by, for example, a factor of 2, 5, 10, or more, in some cases. Fluid components can be chosen by those skilled in the art for particular diffusion or evaporative characteristics. The middle fluid (outer droplet) can also be selected so that the middle fluid provides for transfer of the inner fluid (e.g., containing calcium phosphate precursor) and/or the outer fluid, either into or through the middle fluid. The size (thickness) of the outer droplet may also affect the rate of transfer into and/or out of the inner droplet, and in some cases the thickness of the outer droplet can be selected in order to control the rate at which inner fluid is removed from the inner droplet, or at which an outer fluid swells the inner fluid. Those of ordinary skill in the art will be able to optimize such a system, using no more than routine skill, to achieve a desired diffusion or evaporate a characteristic, depending on the particular application. According to yet another set of embodiments, a specific shell material may be chosen to dissolve, rupture, or otherwise release its contents under certain conditions. For example, if a multiple emulsion contains a calcium phosphate based material, the shell components may be chosen to dissolve under certain physiological conditions (e.g., pH, temperature, osmotic strength), allowing the calcium phosphate based material to be selectively released. If it is desired that the inner species be dried, the shell material may be of a substance that is permeable to water molecules.

A variety of materials and methods, according to certain aspects of the invention, can be used to form any of the above-described components of the systems and devices of the invention, such as, for example, the channels that are used to make multiple emulsions. In some cases, the various materials selected lend themselves to various methods. For example, various components of the invention can be formed from solid materials, in which the channels can be formed via micromachining, film deposition processes such as spin coating and chemical vapor deposition, laser fabrication, photolithographic techniques, etching methods including wet chemical or plasma processes, and the like. See, for example, Scientific American, 248:44-55, 1983 (Angell, et aϊ). In one embodiment, at least a portion of the fluidic system is formed of silicon by etching features in a silicon chip. Technologies for precise and efficient fabrication of various fluidic systems and devices of the invention from silicon are known. In another embodiment, various components of the systems and devices of the invention can be formed of a polymer, for example, an elastomeric polymer such as polydimethylsiloxane ("PDMS"), polytetrafluoroethylene ("PTFE" or Teflon ), or the like.

Different components can be fabricated of different materials. For example, a base portion including a bottom wall and side walls can be fabricated from an opaque material such as silicon or PDMS, and a top portion can be fabricated from a transparent or at least partially transparent material, such as glass or a transparent polymer, for observation and/or control of the fluidic process. Components can be coated so as to expose a desired chemical functionality to fluids that contact interior channel walls, where the base supporting material does not have a precise, desired functionality. For example, components can be fabricated as illustrated, with interior channel walls coated with another material. Material used to fabricate various components of the systems and devices of the invention, e.g., materials used to coat interior walls of fluid channels, may desirably be selected from among those materials that will not adversely affect or be affected by fluid flowing through the fluidic system, e.g., material(s) that is chemically inert in the presence of fluids to be used within the device.

In one embodiment, various components of the invention are fabricated from polymeric and/or flexible and/or elastomeric materials, and can be conveniently formed of a hardenable fluid, facilitating fabrication via molding (e.g. replica molding, injection molding, cast molding, etc.). The hardenable fluid can be essentially any fluid that can be induced to solidify, or that spontaneously solidifies, into a solid capable of containing and/or transporting fluids contemplated for use in and with the fluidic network. In one embodiment, the hardenable fluid comprises a polymeric liquid or a liquid polymeric precursor (i.e. a "prepolymer"). Suitable polymeric liquids can include, for example, thermoplastic polymers, thermoset polymers, or mixture of such polymers heated above their melting point. As another example, a suitable polymeric liquid may include a solution of one or more polymers in a suitable solvent, which solution forms a solid polymeric material upon removal of the solvent, for example, by evaporation. Such polymeric materials, which can be solidified from, for example, a melt state or by solvent evaporation, are well known to those of ordinary skill in the art. A variety of polymeric materials, many of which are elastomeric, are suitable, and are also suitable for forming molds or mold masters, for embodiments where one or both of the mold masters is composed of an elastomeric material. A non-limiting list of examples of such polymers includes polymers of the general classes of silicone polymers, epoxy polymers, and acrylate polymers. Epoxy polymers are characterized by the presence of a three-membered cyclic ether group commonly referred to as an epoxy group, 1,2-epoxide, or oxirane. For example, diglycidyl ethers of bisphenol A can be used, in addition to compounds based on aromatic amine, triazine, and cycloaliphatic backbones. Another example includes the well-known Novolac polymers. Non- limiting examples of silicone elastomers suitable for use according to the invention include those formed from precursors including the chlorosilanes such as methylchlorosilanes, ethylchlorosilanes, phenylchlorosilanes, etc.

Silicone polymers are preferred in one set of embodiments, for example, the silicone elastomer polydimethylsiloxane. Non-limiting examples of PDMS polymers include those sold under the trademark SYLGARD™ by Dow Chemical Co., Midland, MI, and particularly SYLGARD 182™, SYLGARD 184™, and SYLGARD 186™. Silicone polymers including PDMS have several beneficial properties simplifying fabrication of the micro fluidic structures of the invention. For instance, such materials are inexpensive, readily available, and can be solidified from a prepolymeric liquid via curing with heat. For example, PDMSs are typically curable by exposure of the prepolymeric liquid to temperatures of about, for example, about 65°C to about 75°C for exposure times of, for example, about an hour. Also, silicone polymers, such as PDMS, can be elastomeric, and thus may be useful for forming very small features with relatively high aspect ratios, necessary in certain embodiments of the invention. Flexible (e.g., elastomeric) molds or masters can be advantageous in this regard.

One advantage of forming structures such as micro fluidic structures of the invention from silicone polymers, such as PDMS, is the ability of such polymers to be oxidized, for example by exposure to an oxygen-containing plasma such as an air plasma, so that the oxidized structures contain, at their surface, chemical groups capable of cross-linking to other oxidized silicone polymer surfaces or to the oxidized surfaces of a variety of other polymeric and non- polymeric materials. Thus, components can be fabricated and then oxidized and essentially irreversibly sealed to other silicone polymer surfaces, or to the surfaces of other substrates reactive with the oxidized silicone polymer surfaces, without the need for separate adhesives or other sealing means. In most cases, sealing can be completed simply by contacting an oxidized silicone surface to another surface without the need to apply auxiliary pressure to form the seal. That is, the pre-oxidized silicone surface acts as a contact adhesive against suitable mating surfaces. Specifically, in addition to being irreversibly sealable to itself, oxidized silicone such as oxidized PDMS can also be sealed irreversibly to a range of oxidized materials other than itself including, for example, glass, silicon, silicon oxide, quartz, silicon nitride, polyethylene, polystyrene, glassy carbon, and epoxy polymers, which have been oxidized in a similar fashion to the PDMS surface (for example, via exposure to an oxygen-containing plasma). Oxidation and sealing methods useful in the context of the present invention, as well as overall molding techniques, are described in the art, for example, in an article entitled "Rapid Prototyping of Microfluidic Systems and Polydimethylsiloxane," Anal. Chem., 70:474-480, 1998 (Duffy, et al.), incorporated herein by reference.

In some embodiments, certain microfluidic structures of the invention (or interior, fluid- contacting surfaces) may be formed from certain oxidized silicone polymers. Such surfaces may be more hydrophilic than the surface of an elastomeric polymer. Such hydrophilic channel surfaces can thus be more easily filled and wetted with aqueous solutions.

In one set of embodiments, a bottom wall of a microfluidic device of the invention is formed of a material different from one or more side walls or a top wall, or other components. For example, the interior surface of a bottom wall can comprise the surface of a silicon wafer or microchip, or other substrate. Other components can, as described above, be sealed to such alternative substrates. Where it is desired to seal a component comprising a silicone polymer (e.g. PDMS) to a substrate (bottom wall) of different material, the substrate may be selected from the group of materials to which oxidized silicone polymer is able to irreversibly seal (e.g., glass, silicon, silicon oxide, quartz, silicon nitride, polyethylene, polystyrene, epoxy polymers, and glassy carbon surfaces which have been oxidized). Alternatively, other sealing techniques can be used, as would be apparent to those of ordinary skill in the art, including, but not limited to, the use of separate adhesives, thermal bonding, solvent bonding, ultrasonic welding, etc.

The following documents are incorporated herein by reference in their entirety: International Patent Application No. PCT/US2004/010903, filed April 9, 2004, entitled "Formation and Control of Fluidic Species," by Link, et al., published as WO 2004/091763 on October 28, 2004; International Patent Application No. PCT/US2006/007772, filed March 3, 2006, entitled "Method and Apparatus for Forming Multiple Emulsions," by Weitz, et al., published as WO 2006/096571 on September 14, 2006; and U.S. Patent Application Serial No. 12/058,628, filed March 28, 2008, entitled "Emulsions and Techniques for Formation," by Chu, et al., published as U.S. Patent Application Publication No. 2009-0012187 on January 8, 2009. The following working Examples are intended to illustrate certain embodiments of the present invention, but do not exemplify the full scope of the invention, and are not intended to, and should not be construed to limit the full scope of the invention.

EXAMPLE 1 (Overview, and Materials and Methods (e.g., microfluidic emulsification) relating to synthesizing calcium phosphate materials (e.g., mesoporous hydroxyapatite (HAp, (CaJw,

(Pθ4)β(OH)2)) in multiple emulsion (e.g., double emulsion) droplet microreactors)

This example provides an overview and materials and methods discussion relating to an exemplary approach for synthesizing mesoporous hydroxyapatite (HAp, (Ca)₁₀, (PO₄)O(OH)₂) in double emulsion droplet microreactors. By using capillary microfluidic techniques, the size and the geometry of the droplet microreactors can be tuned easily. The droplet microreactors enable relatively simple visualization of the HAp formation process as well as control over the porosity in synthesized HAp. Powder formed with the techniques described herein demonstrates a unique microstructure, as well as significantly enhanced BET specific average surface area and nanoscale porosity. We also demonstrate that amorphous as-processed powders can be transformed to crystalline HAp and tricalcium phosphate (TCP, Ca₃(POz_I)₂) during calcinations at high temperature. These methods can allow for the control of the nanoscale porosity and the morphology of inorganic particles using double emulsion droplets.

The biocompatibility and chemical similarity to bone makes calcium phosphate (CaP) based ceramic materials, such as hydroxyapatite (HAp), a widely used material of choice for orthopedic, dental and other bio-related applications. To mimic the functions of their counterparts in natural biological systems, CaP-based materials can be used in conjunction with different biomolecules to provide surfaces for adsorption and catalysis of biochemical reactions. The adsorption of biomolecules onto the surface of these CaP based materials can depend on their structural properties such as microstructural features, surface area, and porosity. In many previous synthesis methods, agglomeration of final powders cannot be easily controlled. In addition, it can be difficult to produce HAp nanopowder with very high BET surface area, which is advantageous for both densification as well as bone morphogenic protein or drug delivery applications when synthetic CaPs are used in treatments for bone related disorders. Moreover, many conventional methods involve intermediate processing steps that prevent the in-situ visualization of the formation of the inorganic nanoparticles. As a result, the exact mechanism for the formation of CaPs from its precursor materials is still not well understood. In the exemplary examples herein, HAp nanopowder can be fabricated utilizing droplets as reactors, each of which can be monitored individually. The homogeneity of the final powders can be governed by the uniformity of the droplets. By flowing fluids through microchannels of hundreds of microns in size, microfluidic emulsifϊcation offers a means to prepare emulsions with a narrow size distribution by providing a high degree of control over the emulsion generation process. The resulting emulsion droplets can be used as templates for fabricating monodisperse particles, highlighting the versatility and controllability of the technique. Moreover, due to relatively larger droplet size over those with conventional emulsion techniques, subsequent processing of the droplets can be monitored relatively easily using microscopic techniques. Double emulsion droplets may be particularly advantageous as microreactors, in some cases, because droplets are prevented from agglomerating with neighboring droplets by the middle oil shell surrounding the reacting core.

In the exemplary examples herein, HAp has been synthesized using double emulsion droplets as microreactors. A glass capillary micro fluidic set-up was used for generating double emulsion microreactors with calcium and phosphorus precursors encapsulated in the inner aqueous droplet. Formation of HAp can be triggered by adding an alkali to the continuous phase to adjust the pH. Since the alkaline solution was miscible with the continuous phase, no additional homogenization treatment was required. Powders obtained with this technique were shown to exhibit unique microstructures with very high surface area, and mesoporosity. The double emulsion droplet technique provides a way to observe and understand the formation mechanism via direct visualization. The technique is also be used to control morphology and porosity. Materials and methods.

Water-in-oil-in-water (W/O/W) double emulsion droplets. Water-in-oil-in- water (W/O/W) double emulsion droplets were produced using glass microcapillary devices. The inner phase consisted of 0.1-1 M calcium nitrate tetrahydrate (Ca(Nθ3)₂-4H₂θ, >99.0%, SigmaUltra, St. Louis, Missouri, USA) and 0.071-0.71 M phosphoric acid (H₃PO₄, orthophosphoric acid, >85%, Fluka, St. Louis, Missouri, USA) in water. The osmalility of the solutions was measured with a micro osmometer (Advanced Instruments, Inc., Model 3300). Unless otherwise noted, the middle oil phase consisted of 2 wt% surfactant (Dow Corning® 749 fluid, Midland, Michigan, USA) in a silicone oil (Dow Corning® 200 Fluid, 5 cSt, Midland, Michigan, USA). The outer phase was a 5 wt% poly vinyl alcohol aqueous solution (PVA; MW: 13000-23000 g mol^"1, 87-89% hydrolyzed, Aldrich, St. Louis, Missouri, USA). The surfactant stabilized the inner droplets against coalescence with the exterior aqueous phase, while PVA prevented coalescence of the oil droplets. The pH of the double emulsion formed was adjusted by adding 0.1 N ammonium hydroxide solution (NH₄OH, Fluka, St. Louis, Missouri, USA) to the continuous phase. The outer phase was prepared in some runs by dissolving PVA directly in 0.1 N NH₄OH solution to achieve a one-step synthesis of HAp particles. A schematic of the process is shown in FIG. 4. Unless otherwise specified, all other chemicals were obtained from Aldrich. Water with a resistivity of 18.2 MΩ cm^"1 was acquired from a Millipore Milli-Q system (Millipore, Billerica, Massachusetts, USA).

Specifically, Fig. 4 shows a schematic illustration of the double emulsion-templated synthesis of hydroxyapatite. The inner droplets of the double emulsion consist of an aqueous solution of calcium nitrate and phosphoric acid, which are the calcium and the phosphorus precursors. The oil shells surrounding the inner droplets are made up of an inert oil phase. Upon increase of pH by adding ammonium hydroxide to the continuous phase, hydroxyapatite is formed in the inner droplets of the emulsion. Due to their higher osmolality, water diffuses into and swells the inner droplets, leading to an increase in size of the double emulsion droplets and a resulting thinning of the oil shells. Eventually, the oil shells become so thin that the double emulsions destabilize, releasing the hydroxyapatite cores.

Preparation of double emulsion. Monodisperse W/O/W double emulsions were prepared in glass microcapillary devices as shown in FIG. 5 A. (See also Utada, A. S., Lorenceau, E., Link, D. R., Kaplan, P. D., Stone, H. A., & Weitz, D. A. (2005) Science 308, 537-541, which is incorporated herein by reference in its entirety). Fig. 5A(a) shows a schematic of the glass microcapillary device for generating double emulsion droplets. The round capillaries (World Precision Instruments, Inc., Sarasota, Florida), with inner and outer diameters of 0.58 mm and 1.0 mm, respectively, were tapered to desired diameters with a micropipette puller (P-97, Sutter Instrument, Inc.) and a micro forge (Narishige International USA, Inc., East Meadow, New York, USA). Two tapered capillaries were aligned inside square glass capillaries (Altantic International Technology, Inc., Rockaway, New Jersey, USA) with an inner diameter of 1.05 mm. A transparent epoxy resin (5 Minute Epoxy, Devcon, Danvers, Massachusetts, USA) was applied to seal the capillaries where necessary. The outer radii, R₀, of the double emulsions ranged from 40 to 80 microns, while the inner radii, Ri, ranged from 30 to 70 microns. These values were controlled by the size of the capillaries used and the flow rates of the different phases, as described in Utada et al. The calcium and phosphorus precursors were pre-dissolved in the inner phase for subsequent encapsulation in double emulsion droplets. Positive syringe pumps (PHD 2000 series, Harvard Apparatus, Holliston, Massachusetts, USA) were used to deliver the different phases at desired flow rates. A typical set of flow rates for the outer, middle and inner phases was 10, 2.2, and 1.2 mL/h, respectively, and the droplet generation frequency was about 1000 Hz. Samples were collected between a cover slip and a glass slide separated by a 0.5 mm thick silicone isolator. The isolator was pre-filled with 0.1 N ammonium hydroxide solution for pH adjustment. The subsequent formation of HAp was monitored with optical microscopy.

Preparation ofhydroxyapatite (HAp) powders. To prepare HAp powders, a double emulsion with calcium and phosphorus precursors in the inner droplets was collected in vials of 0.1 N ammonium hydroxide solution, which triggered the formation of hydroxyapaptite. After the vials were completely filled, they were capped and sealed with parafilm. The vials were then left tumbling on a tumbler, during which water continuously diffused from the continuous phase to the inner droplets due to an osmotic pressure difference. The double emulsion droplets eventually destabilized, releasing the as formed HAp powders. To wash the as-formed HAp powders, the remaining oil phase in the vials was removed and the aqueous supernatant was replaced with water. After repeating the washing step 5 times, the remaining HAp was dried in an oven at 4O⁰C for 3-7 days to obtain a dry powder for further characterization. The dry powder was characterized both before and after calcination at elevated temperatures.

Direct visualization of HAp formation in double emulsion. Optical microscope images were obtained with 10^χ, 4Ox and 63 ^χ objectives at room temperature using an inverted microscope (DMIRBE, Leica, Wetzlar, Germany), an inverted fluorescence microscope (DMIRB, Leica, Wetzlar, Germany) or an upright fluorescence microscope (DMRX, Leica, Wetzlar, Germany) equipped with a high speed camera (Phantom, V5, V7 or V9, Vision Research, Wayne, New Jersey, USA) or a digital camera (QICAM 12-bit, Qimaging, Surrey, British Columbia, Canada). All double emulsion generation processes were monitored with the microscope using a high speed camera. The formation mechanism of HAp from double emulsions and the resulting HAp powders were imaged using a digital camera.

Characterization of HAp powders . Microstructural analysis: Scanning electron microscope (SEM) images of dried powders were taken using a Zeiss Supra 55VP field emission scanning electron microscope (FESEM, Carl Zeiss, Germany) at an acceleration voltage of 20 kV. Transmission electron microscope (TEM) images were taken on a JEOL JEM-2010 TEM (JEOL, Tokyo, Japan) operated at 200 kV. TEM samples were prepared using a droplet of suspension with high volume fraction of as formed HAp on a 200 or 300 mesh copper grid coated with Lacey carbon (Electron Microscopy Sciences, Hatfϊeld, Pennsylvania, USA). BET specific average surface area analysis: Adsorption and desorption measurements were performed using a Beckman Coulter SA 3100 surface area and pore size analyzer (Beckman Coulter, Fullerton, California, USA) with nitrogen as an adsorbate. The Brunauer- Emmett-Teller (BET) surface areas were calculated fromp/p₀ = 0.3 in the adsorption curve using a BET equation. (See Brunauer, S., Emmett, P. H., & Teller, E. (1938) Journal of the American Chemical Society 60, 309-319, which is incorporated herein by reference) The pore size distributions were calculated from the desorption and the adsorption data using the Barrett- Joyner-Halenda (BJH) model. (See Barrett, E. P., Joyner, L. G., & Halenda, P. P. (1951) Journal of the American Chemical Society 73, 373-380, which is incorporated herein by reference) Prior to each sorption measurement, the sample was out-gassed for Ih at 300 ⁰C under vacuum.

Phase analysis: Thermogravimetric analysis (TGA, Q50, TA Instruments, New Castle, Delaware, USA) was performed to determine the phase transformation temperatures for the as- formed powder. Phase analysis of the powders before and after calcinations were performed by powder X-ray diffraction (XRD) using a Scintag XDS2000 fixed sample position powder diffractometer (Scintag, Cupertino, California, USA) with Cu Ka radiation at 40 kV and 30 mA. The XRD patterns were taken at room temperature in the range of 12°<2Θ< 70° with a scan rate of 1 degree/minute and step size of 0.02 degrees.

EXAMPLE 2

(Monodisperse W/O/W double emulsions droplets were used as reactors where calcium nitrate and phosphoric acid were stored in the inner droplets for the synthesis of HAp)

This working example provides results from synthesis of mesoporous HAp in double emulsions as disclosed herein.

Specifically, monodisperse W/O/W double emulsions droplets were used as reactors where calcium nitrate and phosphoric acid were stored in the inner droplets for the synthesis of HAp. The synthesis was initiated after the addition of ammonium hydroxide according to the following equation:

10 Ca(NO₃)₂ + 6 H₃PO₄ + 20 NH₄OH ==> Ca₁₀(PO₄)₆(OH)₂ + 20 NH₄NO₃+ 18 H₂O [ 1 ]

By adjusting the reactant concentration such that the osmotic pressure was always higher in the inner droplet than in the continuous phase, water could continuously diffuse into the inner droplets. This led to swelling of the inner droplets and a reduction in the thickness of the oil shells. The HAp formed inside the inner droplet was eventually released after the oil shells became too thin to stabilize the double emulsion, as illustrated in the schematic of FIG. 4. The reaction was completed immediately after pH adjustment by adding ammonium hydroxide, as suggested by the instantaneous precipitation shown in FIG. 5B. The increase in pH in the inner droplets was made possible by the addition of ammonium hydroxide, which did not dissociate completely in water. Therefore, the undissociated ammonia molecules could diffuse across the hydrophobic oil shell. The originally transparent inner droplets became turbid as soon as the droplets came into contact with the alkaline solution. No precipitation was observed outside the inner droplets, suggesting that the timescale for the diffusion of the reactants through the oil shells was higher than that for the pH adjustment and precipitation reaction. The behavior was uniform for all observable droplets. The size uniformity of the droplet microreactors enables visualization of multiple precipitation reactions simultaneously. Due to high concentration of the reactants in the inner droplets, the osmolality was typically much higher in the inner droplet. An inner solution with calcium and phosphorus precursor concentrations of 0.5 M and 0.3 M, respectively, had an osmolality of about 1600 mOsm compared to about 100 mOsm for the outer continuous phase. Not wishing to be bound to any particular theory, the resulting osmotic pressure difference may have caused water to diffuse from the continuous phase to the inner droplets, leading to their swelling. The relatively large osmotic pressure difference may also have contributed to the slow diffusion of reactants out of the inner droplets and the fast pH adjustment in the inner droplet due to the addition of ammonium hydroxide in the continuous phase. The solid precipitated structures remained compact without any change in size after swelling, as shown in FIG. 5C. The parts of the precipitates that had been bound by the oil- water interface were freely suspended in the inner aqueous phase, as shown in FIG. 5D. Since the volume of the oil shells, V_skei_h surrounding the inner droplets remained constant, the thickness of the oil shell, /, decreased with the increase in radius of the inner droplet, R, as

/ ⁼ V_shei/4πR² for small shell thickness. The thickness of the oil shell in FIG. 5E decreased from 18 microns to 4.8 microns after 91 h. As the shell got thinner, it became more likely for the double emulsion droplets to destabilize, releasing the precipitates.

Specifically, Fig. 5 shows: (a) a schematic of the glass microcapillary device for generating double emulsion droplets; (b-e) Optical microscope images of double emulsion droplets (b) immediately and (c) 91 hours after addition of 0.1 N ammonium hydroxide solution. The change in pH triggers the formation of hydroxyapatite in the inner droplets of the double emulsion. The hydroxyapatite spans the entire volume of the inner droplets initially. As water diffuses into the inner droplets due to a higher internal osmolality, the hydroxyapatite particles remain roughly the same in size. Optical microscope images of (d) a hydroxyapatite particle formed and (e) oil shells at higher magnification. The thickness of the oil shells has reduced from 18 μm to 4.8 μm after 91 hours. Scale bar is 50 μm for (b), (d) and (e), and 150 μm for (c).

EXAMPLE 3 (The versatility of the technique was further illustrated by tuning the micro and nanoscale porosity of the precipitate structures)

In this working example, the versatility of the technique was further illustrated by the ease in tuning the micro and nanoscale porosity of the precipitate structures, which could be adjusted by changing the concentrations of calcium and phosphorus precursors. The precipitate had an open porous structure when the inner solution with calcium and phosphorus precursor concentrations of 0.1 M and 0.06 M were used, as shown in FIG. 6A. All the individual precipitate sub-particles and their attachment points to one another can be clearly seen. As the calcium precursor concentration was increased to 0.5 M, while maintaining the same calcium to phosphorus ratio, the formed precipitate had a denser structure, as shown in FIG. 6B. Individual sub-particles could not be distinguished anymore except for those near the edge of the precipitate, where the sub-particles span outwards into the solution. As the precursor concentrations were increased further to 1 M and 0.6 M for calcium and phosphorus, respectively, the precipitate structure became highly dense with the lowest level of porosity, as shown in FIG. 6C. . Apart from the porosity, the size of the precipitate formed could also be easily adjusted, by varying the size of the double emulsion droplet reactors, which was controlled with the flow rates and the geometry of the glass capillary micro fluidic devices.

Specifically, Figs. 6(a)-(c) show optical microscope images of hydroxyapatite particles formed from precursor solutions with calcium nitrate and phosphoric acid concentrations of (a) 0.1 M and 0.06 M, (b) 0.5 M and 0.3 M, and (c) 1 M and 0.6 M respectively. The structure of the particles formed becomes more compact as the precursor concentration increases. Scale bar is 50 μm for (a) and (b), and 30 μm for (c).

EXAMPLE 4

(Properties and morphology of HAp powders processed via double emulsion were determined)

In this working example, properties and morphology of HAp powders processed via double emulsion were characterized. Micro structural analysis using SEM and TEM: The powder obtained after washing and drying of the precipitates exhibited unique microstructures with high surface area. After repeated washing of the precipitates with water and then drying at 40 ⁰C, the original spherical macro-structures of the precipitates were not retained in the dried powder and shown in FIG. 7A. At higher magnification, the powder appeared to be made up of spherical sub-particles that formed clusters, shown in FIG. 7B. Each spherical sub-particle appeared to assemble from platelet- like particles connected to each other at the center of the sphere, forming a feathery morphology shown in FIG. 1C

Specifically, Figs. 7(a)-(c) show scanning electron microscope (SEM) images of dried hydroxyapatite powder at different magnifications. The hydroxyapatite formed is made up of smaller spherical sub-particles of about 1 μm in size, as shown in (b). Each spherical sub- particle has an open, feathery morphology shown in (c). The powder in the images was formed from a precursor solution of 0.5 M calcium nitrate and 0.3 M phosphoric acid. Scale bar is 20 μm for (a), 2 μm for (b), and 500 nm for (c). The platelet-like particles were also observed in TEM images, as shown in FIGs. 8 A and

8B. These particles, which had a length of about 50 nm and a typical aspect ratio of about 10 or more, overlapped with each other to form a network as shown in FIG. 8B.

Specifically, Figs. 8(a) and (b) show: (a) transmission electron microscope (TEM) image of hydroxyapatite powder; (b) magnified view of the hairs similar to the ones shown in (a). The microstructure of the powder is dominated by nanometer-scaled sub-particles shown in (b). The powder in the images was formed from a precursor solution of 1 M calcium nitrate and 0.6 M phosphoric acid. Scale bar is 300 nm for (a), and 100 nm for (b).

BET surface area analysis: The unique morphology of the HAp powders synthesized in the double emulsion droplet reactors gave rise to high porosity and surface area. For an inner solution with 1 M and 0.6 M calcium and phosphorus precursor concentrations, respectively, the BET surface area of the powder was 162.8 m²/g, as compared to 26.0 m²/g for powders obtained from bulk reaction. The powders were produced in the absence of any micellar templates or catalysts. The nitrogen sorption isotherms for both powders exhibited hysteresis loops, which are characteristic of mesoporous type IV isotherms, as shown in FIG. 9A. However, the volume adsorbed was consistently higher at all relative pressure for the powder prepared in the double emulsion droplet reactors, which is in agreement with its higher surface area. The total pore volume was 0.499 ml/g for the powder prepared in double emulsion droplet reactors, as compared to 0.133 ml/g for the powder prepared in bulk. Apart from having more pore volume, powder formed in droplets also had a different pore size distribution, as shown in FIG. 9B.

While both powders had a large proportion of pores with diameters between 20 nm and 80 nm, the powder prepared in droplets also had about 20% of pores with diameters under 6 nm.

Specifically, Figs. 9(a) and (b) show: (a) nitrogen adsorption/desorption isotherms and (b) pore size distributions of hydroxyapatite in double emulsion droplet reactors and in bulk. The powder examined was formed from a precursor solution of 1 M calcium nitrate and 0.6 M phosphoric acid.

Phase analysis: For phase and compositional analysis, the powder was heat-treated or calcined at various temperatures. From thermogravimetric analysis (TGA), the powder formed in the droplet reactors first experienced a steady decrease in weight until about 200⁰C due to the evaporation of adsorbed water and other low boiling point contaminants, if any, as shown in FIG. 10.

Specifically Fig. 10 shows thermogravimetric (TG) curves of the hydroxyapatite formed. The sample heating rate was 5 °C/min and the sample was calcined at 500 ⁰C and 1000 ⁰C each for 10 minutes. The inset is a scanning electron microscope (SEM) image of the powder after thermogravimetric analysis. The powder examined was formed from a precursor solution of 1 M calcium nitrate and 0.6 M phosphoric acid. Scale bar in the inset is 5 μm.

At about 250⁰C, the weight dropped at a higher rate until about 300⁰C. The slope of the curve remained almost the same until about 820⁰C where another dip occurred. The weight loss until 820⁰C was attributed to carbon loss from the system, due to the high heating rate of 5⁰C /min. The weight of the sample then started to level off at about 86.5% above 850⁰C. The sudden sharp drop in slope at 500⁰C was due to the 10-minute holding time at the temperature and does not correspond to any change in phase. To study the phase transformations behavior, room temperature XRD was used to analyze the powder after calcinations at various temperatures. XRD patterns for the powder before and after calcinations at 600⁰C appeared very similar to those that show signature peaks for HAp (JCPDS # 09-0432), as shown in the curves labeled a and b in FIG. 11. The peaks were wide indicating that the powder was still partially amorphous, even after calcination at 600⁰C for Ih. The peaks obtained from the powder after calcination at 800⁰C for Ih became significantly sharper, revealing pure crystalline HAp phase, as shown in the curve labeled c in FIG. 11. Interestingly, after the powder had been calcined at 1000⁰C for Ih, more peaks appeared in the powder XRD pattern, as shown in the curve labeled d in FIG. 11. A combination of peaks primarily from both HAp and β-TCP (Ca₃(PO₄)₂) (JCPDS # 09-0169) phases were observed with a small amount of α-TCP (JCPDS # 09-0348). When the powder was calcined at a slower rate of 5°C/min, instead of 30°C/min, the powder was completely converted to β-TCP and CaO (JCPDS # 82-1691), as indicated by the curve labeled e in FIG. 11.

Specifically, Fig. 11 shows: X-ray diffraction (XRD) patterns of hydroxyapatite powder (a) as formed, and calcined at (b) 600⁰C, (c) 800⁰C, and (d) 1000⁰C. Samples were heated at 30°C/min to and then kept at the temperature specified for one hour, (e) XRD pattern of powder heated at 5°C/min to and calcined at 1000⁰C. All powders examined were formed from a precursor solution of 1 M calcium nitrate and 0.6 M phosphoric acid. The squares, stars, circles and pounds designated as β-tricalcium phosphate (TCP), α-tricalcium phosphate, hydroxyapatite and calcium oxide indicate the characteristic peaks of α-TCP (JCPDS # 09-0348, β-TCP (JCPDS file # 09-0169), hydroxyapatite (JCPDS file # 09-0432) and calcium oxide (JCPDS # 82-1691) at the marked positions respectively.

Direct visualization of HAp formation. Direct visualization of HAp formation from its precursors in microreactors was observed. Visualization of HAp formation facilitates characterization and understanding of different stages of formation as well as influence of precursor concentration on the morphology of as formed mesoporous HAp powders. It was shown that lower precursor concentration in the microreactor helped to form first a highly porous, networked, nanostructured HAp, and then with time some consolidation took place in the as formed powders within the microreactor. With increasing precursor concentration within the same size of double emulsion droplet microreactors, the as-formed HAp morphology changed from an open ended network mesoporous structure to a dense mesoporous structure. The total pore volume, 0.499 ml/g, was significantly higher for the powder prepared in double emulsion droplet reactors as compared to the powder prepared in bulk, 0.133 ml/g. While most of the porosity was between 20 nm and 80 nm in diameter for powders synthesized in bulk and in microreactors, the HAp powder prepared in droplet microreactor also has about 20% of pores with diameters under 6 nm. The presence of nanoscale porosity significantly increases the specific average surface area of the as formed powders to 162.8 m²/g, as compared to 26.0 m²/g for powders obtained from bulk reaction. Calcination of as formed powder up to 800⁰C results in the formation of phase pure HAp.

While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present invention is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the scope of the present invention.

The indefinite articles "a" and "an," as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

The phrase "and/or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to "A and/or B," when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e. "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and/or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

In the claims, as well as in the specification above, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of and "consisting essentially of shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

Claims

1. A method of forming a calcium phosphate based material, comprising: surrounding a first fluid with a second fluid; surrounding the second fluid with a third fluid, wherein the first and third fluids are suitably immiscible with the second fluid to provide a multiple emulsion microreactor, and wherein at least one of the first, second and third fluids comprise calcium phosphate precursor; and forming, in at least one fluid of the multiple emulsion microreactor, a calcium phosphate based material from the calcium phosphate precursor, wherein a method of forming a calcium phosphate based material is provided.

2. The method of claim 1, comprising providing to at least one of the first, second and third fluids of the multiple emulsion microreactor contains an additional reagent that facilitates formation of calcium phosphate from calcium phosphate precursor, and wherein the fluid to which the additional reagent is provided is different than the fluid containing the calcium phosphate precursor.

3. The method of claim 2, wherein the additional reagent comprises alkaline material.

4. The method of claim 2, wherein formation of calcium phosphate from calcium phosphate precursor comprises diffusion of the additional reagent into the fluid containing the calcium phosphate precursor.

5. The method of claim 4, wherein at least one of the first and third fluids contains the calcium phosphate precursor, and wherein the additional reagent diffuses from the second fluid into the fluid containing the calcium phosphate precursor.

6. The method of claim 4, wherein the second fluid contains the calcium phosphate precursor, and wherein the additional reagent diffuses from at least one of the first and third fluids into the second fluid containing the calcium phosphate precursor.

7. The method of claim 1, comprising a plurality of double emulsion microreactors, wherein the third fluid comprises a continuous phase contacting said plurality, and wherein the inner, first fluid contains calcium phosphate precursor.

8. The method of claim 7, comprising providing to at least one of the second and third fluids of the double emulsion microreactor an additional reagent that facilitates formation of calcium phosphate from calcium phosphate precursor, and wherein formation of calcium phosphate from calcium phosphate precursor comprises diffusion of the additional reagent into the first fluid containing the calcium phosphate precursor.

9. The method of claim 1 , comprising a plurality of double emulsion microreactors, wherein the third fluid comprises a continuous phase contacting said plurality, and wherein the second fluid contains calcium phosphate precursor.

10. The method of claim 9, comprising providing to at least one of the first and third fluids of the double emulsion microreactor an additional reagent that facilitates formation of calcium phosphate from calcium phosphate precursor, and wherein formation of calcium phosphate from calcium phosphate precursor comprises diffusion of the additional reagent into the second fluid containing the calcium phosphate precursor.

11. The method of claim 1 , wherein the osmolality or osmotic pressure within the fluid containing the calcium phosphate precursor is higher than the osmolality or osmotic pressure of at least one of the other fluids.

12. The method of claim 1 , comprising a plurality of double emulsion microreactors, wherein the third fluid comprises a continuous phase contacting said plurality, wherein the first fluid contains calcium phosphate precursor, the method comprising providing to the third continuous phase fluid an additional reagent that facilitates formation of calcium phosphate from calcium phosphate precursor, and wherein formation of calcium phosphate from calcium phosphate precursor comprises diffusion of the additional reagent into and through the second fluid and in turn into the fist fluid containing the calcium phosphate precursor.

13. The method of claim 12, wherein the first and third fluids are aqueous based, wherein the second fluid is oil based, and wherein the osmolality or osmotic pressure within the first fluid containing the calcium phosphate precursor is higher than the osmolality or osmotic pressure of the third fluid.

14. The method of claim 12, wherein the additional reagent comprises alkaline material.

15. The method of claim 1, wherein at least one of the fluids contains, or receives an alkaline material.

16. The method of claim 1, comprising, during formation of the calcium phosphate based material, increasing the volume occupied by the fluid containing the calcium phosphate precursor.

17. The method of claim 1, wherein the multiple emulsion microreactor is a droplet multiple emulsion microreactor.

18. The method of claim 1 , wherein the calcium phosphate based material comprises or consists essentially of at least one of hydroxyapatite and tricalcium phosphate, and β-tricalcium phosphate.

19. The method of claim 1 , wherein the morphology of the calcium phosphate based material comprises at least one of monodisperse, crystalline, mesoporous, nanoporous, needle-like nanoparticles, and microspheres comprising needle-like nanoparticles.

20. The method of claim 1 , wherein the calcium phosphate based material comprises at least one of: a portion with an aspect ratio of at least about 5 : 1 with a minor dimension defining the aspect ratio of less than about 1 micron; nanoscale pores; porosity wherein at least about 10 vol% of the pores have diameters of less than about 6 nm; porosity wherein at least about 20 vol% of the pores have diameters of less than about 6 nm; a BET surface area of at least about 150 m²/g; a total pore volume of at least about 0.25 mL/g; a total pore volume of at least about 0.50 mL/g; and a Ca to P ratio, by atomic number, of between about 1 : 1 and about 2:1.

21. The method of claim 1 , wherein the calcium phosphate material at least partially encapsulates or attaches to another material.

22. The method of claim 1 , further comprising heat treating or calcining the calcium phosphate based material.

23. A composition or article comprising a calcium phosphate based material prepared according to the method of any one of claims 1 through 22.

24. A composition comprising a calcium phosphate based material, wherein the calcium phosphate based material comprises at least one of: a portion with an aspect ratio of at least about 5 : 1 with a minor dimension defining the aspect ratio of less than about 1 micron; nanoscale pores; porosity wherein at least about 10 vol% of the pores have diameters of less than about 6 nm; porosity wherein at least about 20 vol% of the pores have diameters of less than about 6 nm; a BET surface area of at least about 150 m²/g; a total pore volume of at least about 0.25 mL/g; a total pore volume of at least about 0.50 mL/g; and a Ca to P ratio, by atomic number, of between about 1 : 1 and about 2: 1.

25. The composition of claim 24, wherein the calcium phosphate based material comprises or consists essentially of at least one of hydroxyapatite and tricalcium phosphate, and β-tricalcium phosphate.

26. The composition of claim 24, wherein the calcium phosphate material at least partially encapsulates or attaches to another material.

27. The composition of claim 24, comprising heat-treated or calcined calcium phosphate based material.