WO2009110917A1 - Bone cement compositions for use as growth factor carriers and methods of making same - Google Patents

Abstract

A self-hardening bone replacement material and may include a combination of a calcium phosphate cement composition, a physiologically acceptable setting solution, a macropore forming powder, and at least protein growth factor. The bone replacement material may be hardenable in vivo. A macropore forming powder may be capable of dissolving in vivo at a known dissolution rate, such that macropores are formed in the matrix of the bone replacement material, thereby facilitating ingrowth of natural bone therein. The macropore forming powder may further be capable of functioning as an excipient, thereby facilitating the release of the growth factor to the surrounding tissue microenvironment.

Description

BONE CEMENT COMPOSITIONS FOR USE AS GROWTH FACTOR CARRIERS

AND METHODS OF MAKING SAME

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention generally relates to calcium phosphate cement compositions that are suitable for use as pharmaceutical carriers, and to methods for making same.

2. Description of the Relevant Art

Defects in bone structure arise in a variety of circumstances, such as trauma, disease, and surgery. There is a need for effective repair of bone defects in various surgical fields, including maxillo-craniofacial, periodontics, and orthopedics. Numerous natural and synthetic materials and compositions have been used to stimulate healing at the site of a bone defect. As with compositions used to repair other types of tissue, the biological and mechanical properties of a bone repair material are critical in determining the effectiveness and suitability of the material in any particular application.

After blood, bone is the second most commonly transplanted material. Autologous cancellous bone has long been considered the most effective bone repair material, since it is both osteoinductive and non-immunogenic. However, adequate quantities of autologous cancellous bone are not available under all circumstances, and donor site morbidity and trauma are serious drawbacks to this approach. The use of allograft bone avoids the problem of creating a second surgical site in the patient, but suffers from some disadvantages of its own. For instance, allograft bone typically has a lower osteogenic capacity than autograft bone, a higher resorption rate, creates less revascularization at the site of the bone defect, and typically results in a greater immunogenic response. The transfer of certain diseases is also a danger when using allografts.

To avoid the problems associated with autograft and allograft bone, considerable research has been conducted in the area of synthetic bone substitute materials that can be used in lieu of natural bone. For example, various compositions and materials comprising demineralized bone matrix, calcium phosphate, and calcium sulfate have been proposed.

Cements comprising calcium phosphates have a long history of use as bone graft substitutes. Modern surgical grade calcium phosphate cements offer high initial strength, good handling properties, and are consistently replaced by bone in many applications. However, calcium sulfate cements are characterized by relatively rapid resorption by the body, which can be undesirable in certain applications.

Hydroxyapatite is one of the most commonly used calcium phosphates in bone graft materials. Its structure is similar to the mineral phase of bone and it exhibits excellent biocompatibility. However, hydroxyapatite has an extremely slow resorption rate that may be unsuitable in certain applications. Other calcium phosphate materials have also been used in the art, such as β-tricalcium phosphate, which exhibits a faster resorption rate than hydroxyapatite, but has less mechanical strength. Certain calcium phosphate materials that set in situ have also been attempted, such as mixtures of tetracalcium phosphate and dicalcium phosphate anhydrate or dihydrate, which react to form hydroxyapatite when mixed with an aqueous solution. The presently available synthetic bone repair materials do not present ideal functional characteristics for all bone graft applications. As noted above, some compositions exhibit a resorption rate that is either too slow or too rapid. Further, many bone graft cements are difficult to implant because they fail to set or cannot be injected. Other drawbacks are inadequate strength and difficulty in adding biologically active substances for controlled release. For these reasons, there remains a need in the art for bone substitute cement compositions that combine a desirable resorption rate with high mechanical strength, ease of handling, and osteoconductivity.

SUMMARY OF THE INVENTION

A self-hardening cementitious bone substitute composition may include calcium phosphate cement particles, at least one excipient, a physiologically acceptable aqueous setting solution, and at least one polypeptide growth factor. The growth factor may be BMP-2.

A bone substitute composition may include: a self-hardening cementitious composition comprising: a calcium phosphate matrix; at least one macropore forming component, wherein at least a portion of the particles of said component are dispersed throughout said matrix; and at least one polypeptide growth factor; wherein said macropore forming component is capable of functioning as an excipient and is selected such that the bone substitute composition releases more of the polypeptide growth factor into an aqueous medium than an identical bone substitute composition which lacks the macropore forming component.

A bone substitute composition may include a self-hardening cementitious composition comprising: a calcium phosphate matrix; at least one macropore forming component, wherein at least a portion of the particles of said powder are dispersed throughout said matrix; and at least one polypeptide growth factor; wherein said macropore forming component is capable of functioning as an excipient and is selected such that the bone substitute composition, when immersed in an aqueous medium for about 10 days, releases about 4 to about 35 fold more of the polypeptide growth factor into the aqueous medium than an identical bone substitute composition which lacks the macropore forming component.

A bone substitute composition may include: a self -hardening cementitious composition comprising: a calcium phosphate matrix; at least one macropore forming component, wherein at least a portion of the particles of said component are dispersed throughout said matrix; and at least one polypeptide growth factor; wherein at least about 4% of the growth factor is released from the bone substitute composition when immersed in an aqueous medium for 10 days.

BRIEF DESCRIPTION OF THE DRAWINGS

The above brief description as well as further objects, features and advantages of the methods and apparatus of the present invention will be more fully appreciated by reference to the following detailed description of presently preferred but nonetheless illustrative embodiments in accordance with the present invention when taken in conjunction with the accompanying drawings in which:

FIG. 1 is a depiction of a representative rhBMP-2 release profile from a control CPC, and a CPC with excipient A. rhBMP-2 was present in the CPC setting solution;

FIG. 2 is a depiction of a representative BMP-2 release profiles from a CPC with excipient G. rhBMP-2 was mixed with the setting solution;

FIG. 3 is a depiction of a representative of a BMP-2 release profile from various CPC/Porogen compositions having 2:1 (C, D and D+F), 3:2 (H) or 2:0.5 (B and I+G) cement:porogen ratio. BMP-2 was mixed with the setting solution;

FIG. 4 is a depiction of a representative BMP-2 release profile from polymer/CPC mixture with 10%PLA; 5% PGA, PLA-PEO (0.04 g/mL in 0.7 M phosphate setting solution) cement;

FIG. 5 is a depiction of a representative BMP-2 release profile from a CPC in which 103 μL of reconstituted rhBMP-2 solution from the rhBMP-2 vial at a concentration of 4mg/ml was used as setting solution and mixed with 0.5 g CPC Carrier directly;

FIG. 6 is a depiction of a representative BMP-2 release profile from a CPC in which 3 mL reconstituted rhBMP-2 solution at 1.5mg/ml were directly used as setting solutions and mixed with 15 g Osteofix Carrier directly; FIG. 7 shows the cytotoxicity of a self-setting CPC carrier with excipient D and H;

FIG. 8 shows the cytotoxicity of a self-setting CPC carrier having the indicated excipient with and without BMP-2;

FIG. 9 shows the biological activity of BMP-2 extracted from a self-setting CPC carrier treated with extracting medium. (BMP-2 release profiles of the same cements are shown in Figure 5);

FIG. 10 is a representative depiction of MC3T3-E1 (subclone 4) cells cultured on a BMP-2-loaded self- setting CPC carrier for 3 weeks. The SEM images show collagen formation and new hydroxyapatite mineralization on the collagen matrix. Alizarin Red staining also indicates new HA formation on the cultures in the presence of the BMP-2-loaded carrier;

FIG. 11 shows representative SEM images of self setting CPC carrier control and self setting CPC carrier with excipients H+L; and

FIG. 12 shows the mechanical strength CPC carrier formulation H + I, upon compression.

While the invention is susceptible to various modifications and alternative forms, specific embodiments thereof are shown by way of example in the drawings and will herein be described in detail. It should be understood, however, that the drawing and detailed description thereto are not intended to limit the invention to the particular form disclosed, but on the contrary, the intention is to cover all modifications, equivalents and alternatives falling within the spirit and scope of the present invention as defined by the appended claims.

DETAILED DESCRIPTION Definitions:

It is to be understood that the present invention is not limited to particular devices or biological systems, which may, of course, vary. It is also to be understood that, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include singular and plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a linker" includes one or more linkers. It is to be yet further understood that any terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

The terms used throughout this specification generally have their ordinary meanings in the art, within the context of the invention, and in the specific context where each term is used. Certain terms are discussed below or elsewhere in the specification to provide additional guidance to the practitioner in describing the general embodiments of the invention, as well as how to make and use them. It will be readily appreciated that the same thing can be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein, nor is any special significance to be placed upon whether or not a term is elaborated or discussed in greater detail herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification, including examples of any terms discussed herein, is illustrative only, and in no way limits the scope and meaning of the invention or of any exemplified term. As used herein, the term "bone substitute material" generally refers to any biocompatible composition, including but not limited to natural bone graft, particulate or crushed bone, demineralized bone matrix, ceramics, polymer systems, composites, or mixtures thereof, that is suitable for use in medical/dental applications characterized by replacement, augmentation, or filling of a hardened calcified tissue (e.g., bone, teeth, enamel and the like). In the context of certain descriptions set forth herein, the term refers to calcium phosphate-based ceramic materials (such as cements) that form hardened structures having physical and chemical properties approximating those of natural bone mineral.

As used herein, the term calcium phosphate cement (CPC) generally refers to a biocompatible material having particles of at least calcium phosphate ceramic material which, when combined with a suitable setting liquid forms a self-setting composition that hardens to become calcium phosphate-based solid mass. In certain embodiments, CPC particles may have an average diameter in the range of about 0.05 μm to about 500 μm, in the range of about 0.1 μm to about 50 μm, or in the range of about 0.5 μm to about 10 μm. A variety of CPCs are known in the art and detailed descriptions thereof, as well as their method of manufacture may be found, for example, in the disclosures of U.S. Patent Nos. 7,018,460; 6,994,726; 6,972,130; 6,960,249;

6,955,716; 6,953,594; 6,929,692; 6,916,177; 6,908,506; 6,905,516; 6,855,167; 6,849,275;

6,840,995; 6,821,528; 6,808,561; 6,796,378; 6,793,725; 6,777,001; 6,730,324; 6,719,993;

6,719,773; 6,716,216; 6,706,273; 6,706,067; 6,703,038; 6,692,563; 6,670,293; 6,648,960;

6,642,285; 6,641,587; 6,620,236; 6,616,742; 6,613,054; 6,599,516; 6,596,338; 6,593,394; 6,592,513; 6,582,228; 6,558,709; 6,547,866; 6,541,037; 6,537,589; 6,527,810; 6,495,156;

6,417,247; 6,384,197; 6,379,453; 6,375,935; 6,338,752; 6,325,992; 6,296,667; 6,241,734;

6,224,629; 6,214,368; 6,206,957; 6,149,655; 6,139,578; 6,136,029; 6,117,456; 6,083,229;

6,053,970; 6,051,061; 6,027,742; 6,018,095; 6,005,162; 6,002,065; 5,997,624; 5,993,535;

5,980,625; 5,976,234; 5,968,253; 5,964,932; 5,962,028; 5,954,867; 5,952,010; 5,900,254; 5,891,558; 5,885,540; 5,866,155; 5,846,312; 5,820,632; 5,782,971; 5,709,742; 5,697,981;

5,695,729; 5,683,667; 5,683,496; 5,679,294; 5,605,713; 5,571,493; 5,569,442; 5,545,254;

5,542,973; 5,534,244; 5,525,148; 5,522,893; 5,503,164; 5,496,399; 5,460,803; 5,437,857;

5,427,768; 5,338,356; 5,336,264; 5,281,265; 5,262,166; 5,218,035; 5,178,845; 5,152,836; 5,149,368; 5,053,212; and 4,373,217, all of which are expressly incorporated herein by reference. In some embodiments, CPC particles may refer to particles of one or more of alpha- tricalcium phosphate (α-TCP), beta-tricalcium phosphate (β-TCP), tetracalcium phosphate (TTCP), monocalcium phosphate monohydrate (MCPM), monocalcium phosphate anhydrous (MCPA), dicalcium phosphate dihydrate (DCPD), dicalcium phosphate anhydrous (DCPA), octacalcium phosphate (OCP), calcium dihydrogen phosphate, calcium dihydrogen phosphate hydrate, acid calcium pyrophosphate, anhydrous calcium hydrogen phosphate, calcium hydrogen phosphate hydrate, calcium pyrophosphate, calcium triphosphate, calcium phosphate tribasic, calcium polyphosphate, calcium metaphosphate, anhydrous tricalcium phosphate, tricalcium phosphate hydrate, and amorphous calcium phosphate.

In some embodiments, the hardened material formed using CPC particles (which may be referred to a "hardened CPC") may be substantially composed of an apatite material. Hydroxyapatite CPCs may optionally include one or more additives that affect the physico- chemical properties of hardened cements made therewith. Exemplary though non-limiting additives which may find use in certain embodiments include those additives selected from the group consisting of sodium phosphate (Na₃PO₄), disodium hydrogen phosphate (Na₂HPO₄), sodium dihydrogen phosphate (NaH₂PO₄), disodium hydrogen phosphate dodecahydrate (Na₂HPO₄- 12H₂O), disodium hydrogen phosphate heptahydrate (Na₂HPO₄-7H₂0), sodium phosphate dodecahydrate (Na₃PO4 12H₂O), orthophosphoric acid (H₃PO₄), calcium sulfate (CaSO₄), Ca₄(PO₄)₂O, CaHPO₄-2H₂O, CaHPO₄, Ca₈H₂(PO₄)₆-5H₂O, alpha-Ca₃(PO₄)₂, beta- Ca₃(PO₄)₂, Ca₂P₂O₇, and Ca₂H₂P₂O₈, (NH₄)₃PO₄, (NH₄)₂HPO₄, and (NH₄)H₂PO₄.

The terms "setting liquid," "setting solution," and the like generally refer to aqueous solutions that, when contacted with CPC particles to form a cementitious mixture, allow cementing reactions to occur between said particles. Exemplary setting liquids suitable for use in the presently disclosed embodiments include, though are not limited to, acidic solutions, a basic solutions, solutions having substantially physiological pH (e.g., 6.0-8.5), including autologous tissues, such as blood or marrow and their derivatives, or substantially pure water. Suitable acidic solutions may include solutions containing nitric acid (HNO₃), hydrochloric acid (HCl), phosphoric acid (H₃PO₄), carbonic acid (H₂CO₃), sodium dihydrogen phosphate (NaH₂PO₄), sodium dihydrogen phosphate monohydrate (NaH₂PO4»H₂O), sodium dihydrogen phosphate dihydrate, sodium dihydrogen phosphate dehydrate, potassium dihydrogen phosphate (KH₂PO₄), ammonium dihydrogen phosphate (NH₄H₂PO₄), malic acid, acetic acid, lactic acid, citric acid, malonic acid, succinic acid, glutaric acid, tartaric acid, oxalic acid and their mixture. Suitable basic solutions may include solutions containing ammonia, ammonium hydroxide, alkali metal hydroxide, alkali earth hydroxide, disodium hydrogen phosphate (Na₂HPO₄), disodium hydrogen phosphate dodecahydrate, disodium hydrogen phosphate heptahydrate, sodium phosphate dodecahydrate (Na₃PO₄* 12H₂O), dipotassium hydrogen phosphate (K₂HPO₄), potassium hydrogen phosphate trihydrate (K₂HPO₄*3H₂O), potassium phosphate tribasic (K₃PO₄), diammonium hydrogen phosphate ((NH₄)₂HPO₄), ammonium phosphate trihydrate ((NH₄)₃PO₄*3H₂O), sodium hydrogen carbonate (NaHCO₃), sodium carbonate Na2CO₃, and their mixture. Non-limiting examples of solutions having substantially physiological pH (i.e., a pH in the range of about 6.5 to about 8.5) include solutions containing phosphate ions such as, e.g., a phosphate buffer, phosphate buffered saline (PBS), Hank's solutions, serum or complex solutions of biomolecules (e.g., a mixture of various serum and/or marrow protein in a physiologically buffered aqueous medium), reaming irrigation fluid, marrow wash, bone suspensions, bone chips and the like. Alternatively, a setting liquid may refer to an aqueous solution suitable for reconstituting a desiccated or otherwise preserved protein or polypeptide mixture. For example, certain embodiments of the present invention are directed to cementitious compositions that include one or more recombinant polypeptide growth factors dispersed therein. In such cases, a setting liquid having a composition suitable for reconstituting recombinant growth factor may be utilized. By way of non-limiting example, in embodiments in which one or more BMP proteins are utilized, the solution in which the recombinant protein preparation is to be reconstituted may itself be used as a setting liquid. In the case of BMPs, such a solution may generally include a buffered aqueous preparation having a pH in the range of about 3.5 to about 5.5, or in the range of about 4.0 to about 7.0, and containing from about 0.05 to about 5 wt.% sucrose, from about 0.5 to about 50 mM glutamate, from about 0.001 vol.% to about 0.1 vol.% polysorbate-80, from about 0.25 wt.% to about 25 wt.% glycine, and from about 0.5 to about 50 mM NaCl. Of course, it will be readily understood by the skilled practitioner that one or more of the above mentioned components may be absent from a final preparation, or that one or more components may be substituted for alternative components, without departing from the sprit and scope of the present disclosure. Likewise, it will be understood that a multitude of other reconstituting buffers may be substituted for that set forth above. Thus, the above is not intended to limit the types of protein reconstitution buffers that may be suitable for use in the presently described embodiments, but rather, the intent is to demonstrate to the skilled practitioner a representative buffer that is suitable for reconstituting a recombinant protein as well as use herein as a suitable setting buffer. Likewise, it will be understood by the skilled practitioner that the presence or absence of a recombinant protein in a setting solution will have no appreciable effect on the suitability of a given reconstitution buffer as a setting liquid (i.e., a buffer having a recombinant protein dissolved therein will generally have the same or similar properties when used as a setting solution as the identical buffer lacking the recombinant protein. The term "self-hardening calcium phosphate cement" generally refers to a composition resulting contacting calcium phosphate cement particles with a setting solution to produce a viscous cementitious paste that is capable of undergoing a cementing reaction. A calcium phosphate cement is said to be "injectable" in the present context when the CPC particles and the setting liquid are combined in a ratio such that the resulting mixtures is sufficiently viscous to allow the injection thereof through a standard medical syringe. Such a mixture may typically be obtained by mixing the CPC particles and the setting solution in a ratio of about 0.1 g/ml to about 20 g/ml. Non-limiting examples of self-setting calcium phosphate cement pastes suitable for use herein are described in U.S. patent application No. 20030216777 entitled "Method of enhancing healing of interfacial gap between bone and tendon or ligament," U.S. patent application No. 20040031420 entitled "Calcium phosphate cement, use and preparation thereof," U.S. patent application No. 20040175320 entitled "Tetracalcium phosphate (TTCP) having calcium phosphate whisker on surface and process for preparing the same," U.S. patent application No. 20050069479 entitled "Method of increasing working time of tetracalcium phosphate cement paste," U.S. patent application No. 20050101964 entitled "Spinal fusion procedure using an injectable bone substitute," U.S. patent application Nos. 20050271740,

20050271741, 20050271742 and 20050268819 entitled "Injectable calcium phosphate cements and the preparation and use thereof," U.S. patent No. 6960249 and U.S. patent application Nos. 20050268820 and 20050279252, U.S. patent application No. 20040003757 entitled "Tetracalcium phosphate (TTCP) having calcium phosphate whisker on surface," U.S. patent application No. 20050268821 entitled "Tetracalcium phosphate (TTCP) with surface whiskers and method of making same," U.S. patent application Nos. 20050274282, 20050274286 and 20050274287 entitled "Calcium phosphate cements made from (TTCP) with surface whiskers and process for preparing same," U.S. patent application Nos. 20050274288 20050279256 20050274289 20060011099 20060011100 entitled "Process for affecting the setting and working time of bioresorbable calcium phosphate cements," U.S. patent Nos. 6379453, 6840995 and U.S. patent application No. 20030121450 entitled "Process for producing fast-setting, bioresorbable calcium phosphate cements," U.S. patent No. 6616742 and U.S. patent application No. 20030078317 entitled "Process for preparing a paste from calcium phosphate cement," U.S. patent No. 6648960 entitled "Method of shortening a working and setting time of a calcium phosphate cement (CPC) paste," U.S. patent No. 6994726 and U.S. patent application No. 20050267592 entitled "Dual function prosthetic bone implant and method for preparing the same," U.S. patent application No. 20050184417 entitled "Method for making a porous calcium phosphate article," U.S. patent application No. 20050029701 entitled "Method for making a molded calcium phosphate article," all of which are commonly owed with the present invention, and the entire contents of which are expressly incorporated by reference as though fully set forth herein.

The terms "non-dispersive," "non-dispersible," and the like, when used in the context of the presently described calcium phosphate cements, generally refers to a physical property of the cement whereby a paste made by combining the cement powder with a setting liquid resists dispersion in an aqueous environment. The ability of a calcium phosphate cement paste to resist dispersion may be related to the surface structure of its constituent particles.

The term "cohesiveness" as used herein is a relative term used to describe the appearance of a calcium phosphate cement during the mixing of the cement powder with the setting liquid.

As used herein, the term "whisker," when used in the context of a calcium phosphate material, generally refers to thin, needle-like calcium phosphate crystals that form on the surface of calcium phosphate particles after subjecting the particles to specific processes as defined in the following United States patents and published applications, all of which are commonly owned with the present invention and incorporated herein by reference in their entirety: U.S. patent application No. 20040031420 entitled "Calcium phosphate cement, use and preparation thereof U.S. patent application No. 20040175320 entitled "Tetracalcium phosphate (TTCP) having calcium phosphate whisker on surface and process for preparing the same" U.S. patent application No. 20050069479 entitled "Method of increasing working time of tetracalcium phosphate cement paste" U.S. patent application No. 20050101964 entitled "Spinal fusion procedure using an injectable bone substitute" U.S. patent application Nos. 20050271740, 20050271741, 20050271742 and 20050268819 entitled "Injectable calcium phosphate cements and the preparation and use thereof U.S. patent No. 6960249 and U.S. patent application Nos. 20050268820 and 20050279252 U.S. patent application No. 20040003757 entitled "Tetracalcium phosphate (TTCP) having calcium phosphate whisker on surface" U.S. patent application No. 20050268821 entitled "Tetracalcium phosphate (TTCP) with surface whiskers and method of making same" U.S. patent application Nos. 20050274282, 20050274286 and 20050274287 entitled "Calcium phosphate cements made from (TTCP) with surface whiskers and process for preparing same" U.S. patent application Nos. 20050274288, 20050279256, 20050274289, 20060011099, 20060011100 entitled "Process for affecting the setting and working time of bioresorbable calcium phosphate cements" U.S. patent Nos. 6379453, 6840995 and U.S. patent application No. 20030121450 entitled "Process for producing fast-setting, bioresorbable calcium phosphate cements" U.S. patent No. 6616742 and U.S. patent application No. 20030078317 entitled "Process for preparing a paste from calcium phosphate cement" U.S. patent No. 6648960 entitled "Method of shortening a working and setting time of a calcium phosphate cement (CPC) paste." Additional methods for forming such whiskers on the surface of CPC particles are set forth below. As used herein, the term "porogen" generally refers to any particulate non-toxic biocompatible material that may be incorporated into a CPC formulation and that, upon hardening of said CPC formulation to a hardened CPC, is gradually removed from the hardened CPC matrix (by virtue of its bioresorbability, biodegradability, solubility etc.) leaving a void therein. A porogen may have any 3-dimensional shape, e.g., substantially spherical, substantially cuboidal, substantially cylindrical, substantially pyramidal, substantially ovoid, needle-like crystals with aspect ratio from 1 to 30, or irregular in shape. Typically, a dimension of a porogen will be in the range of about 0.01 μm to about 30 mm. Porogens suitable for use in the present embodiments include crystalline materials (e.g., salts), polypeptides, and polymer compositions. In some instances, the terms "porogen" and "macropore forming powder" may be used interchangeably.

As used herein, the term "whisker," when used in the context of a calcium phosphate material, generally refers to thin, needle-like calcium phosphate crystals that form on the surface of calcium phosphate particles after subjecting the particles to specific processes as defined in the following United States patents and published applications, all of which are commonly owned with the present invention and incorporated herein by reference in their entirety: U.S. patent application No. 20040031420 entitled "Calcium phosphate cement, use and preparation thereof U.S. patent application No. 20040175320 entitled "Tetracalcium phosphate (TTCP) having calcium phosphate whisker on surface and process for preparing the same" U.S. patent application No. 20050069479 entitled "Method of increasing working time of tetracalcium phosphate cement paste" U.S. patent application No. 20050101964 entitled "Spinal fusion procedure using an injectable bone substitute" U.S. patent application Nos. 20050271740, 20050271741, 20050271742 and 20050268819 entitled "Injectable calcium phosphate cements and the preparation and use thereof U.S. patent No. 6960249 and U.S. patent application Nos. 20050268820 and 20050279252 U.S. patent application No. 20040003757 entitled "Tetracalcium phosphate (TTCP) having calcium phosphate whisker on surface" U.S. patent application No. 20050268821 entitled "Tetracalcium phosphate (TTCP) with surface whiskers and method of making same" U.S. patent application Nos. 20050274282, 20050274286 and 20050274287 entitled "Calcium phosphate cements made from (TTCP) with surface whiskers and process for preparing same" U.S. patent application Nos. 20050274288, 20050279256 20050274289 20060011099 20060011100 entitled "Process for affecting the setting and working time of bioresorbable calcium phosphate cements" U.S. patent Nos. 6379453 6840995 and U.S. patent application No. 20030121450 entitled "Process for producing fast-setting, bioresorbable calcium phosphate cements" U.S. patent No. 6616742 and U.S. patent application No.

20030078317 entitled "Process for preparing a paste from calcium phosphate cement" U.S. patent No. 6648960 entitled "Method of shortening a working and setting time of a calcium phosphate cement (CPC) paste."

As used herein, the term "unsintered," when used in the context of the subject prosthetic bone implants, generally refers to a prosthetic bone implant that is made from a hardened calcium phosphate cement and that has not undergone a high temperature sintering step. While sintered calcium phosphate ceramics exhibit relatively high tensile strength and biocompatibility, they typically are less porous, and as a result are generally not bioresorbable. Thus, unsintered calcium phosphate cement articles retain their porosity, and are therefore more bioresorbable than sintered calcium phosphate ceramics. Included within the term "unsintered" are those bioresorbable calcium phosphate articles or cements that have been treated at a temperature up to 75O⁰C, up to 500⁰C, up to 200⁰C, or up to 5O⁰C.

The term "macroporous" is used in reference to a porous material that has cavities or pores whose size (i.e., average diameter) is in the μm range. While it will be readily appreciated by the skilled practitioner that the meaning of the term is somewhat subjective, for the purposes of the present disclosure, the term "macroporous" is meant to refer to a porous material whose pores are generally from about 1 μm to about 10000 μm in diameter. The related term "interconnected porosity" refers to pores or cavities in the body or matrix of the subject prosthetic bone implants that are coupled to each other and in fluid communication so as to form a continuous network of pores capable of conveying liquids or gases, or materials dissolved therein. Typically, the degree of interconnected porosity of a calcium phosphate implant is related to its bioresorbability, wicking potential and ability to promote bone ingrowth.

The term "macropore forming component" is used herein to refer to a composition or substance that has a known dissolution rate in vivo and that, when dispersed in a solid matrix that is implanted in vivo, dissolves leaving macroporous cavities within the matrix. The average diameter of constituent particles of such a powder will generally be in the range of about 1 μm to about 5000 μm or larger. The average length will be in the range of 0.1 to 30mm. As used herein, the term "nanoporous" generally refers to a porous material (i.e. a calcium phosphate ceramic) whose average pore diameter is in the nanometer range (typically between 1 to 1000 nm).

As used herein, the term "apatite" generally refers to a group of phosphate minerals, (e.g., hydroxyapatite, fluorapatite, and chlorapatite) having the general chemical formula Cas(PO₄)₃X, where X is OH, F, or Cl. The term "hydroxyapatite," sometimes referred to as "HA" or "HAp," as used herein generally refers to a form of apatite with the formula Ca_S(PO₄)_S(OH), but is more typically represented as Ca₁₀(PO₄)_O(OH)₂ to denote that the crystal unit cell comprises two molecules. Hydroxyapatite is the hydroxylated member of the complex apatite group. The hardness of hydroxyapatite may be altered by replacing the OH ion with other ions (e.g., fluoride, chloride or carbonate). Additionally, HAp has a relatively high affinity for peptides, making it an ideal carrier for the delivery and/or sustained release of polypeptides over long periods of time in situ. Materials that are refered to herein as "apatitic," are generally those materials that have apatite as the major phase (i.e., materials that are substantially comprised of apatite). As used herein, terms such as "therapeutic composition," generally refers to compositions or agents that are capable of inducing or affecting a biological response action in a biological system, e.g. by inducing or affecting a therapeutic or prophylactic effect, an immune response, tissue growth, cell growth, cell differentiation, cell proliferation, etc. A therapeutic composition/agent may be provided by means of a suitable pharmaceutical delivery vehicle. The delivery vehicle would typically be optimized to stably accommodate an effective dosage of one or more compounds having biological activity. The determination of the effective dose of a bioactive compound that should be included in a therapeutic composition to achieve a desired biological response is dependent on the particular compound, the magnitude of the desired response, and the physiological context of the composition. Such determinations may be readily made by an ordinary practitioner of the pharmaceutical arts. Components of therapeutic compositions may include growth factors, bone proteins, analgesics, antibiotics, or other pharmacologically active compounds.

As used herein, a composition that is referred to as being "physiologically acceptable" is a composition that is non-toxic, biocompatible and whose physical and chemical features (e.g., pH, osmolality, temperature, and the like) fall within a range that is substantially unlikely to induce or be the cause of adverse physiological responses (e.g., inflammation, hypersensitivity, toxicity, and the like). A "physiologically acceptable" aqueous solution will typically have a pH in the range of about 6.0 to about 8.5, in the range of about 7.0 to 8.0, or in the range of about 7.2 to about 7.6. Such a solution will typically have an osmolarity in the range of about 200 to about 500 mOsmol/L, about 250 to about 350 mOsmol/L or about 280 to about 310 mOsmol/L. Thus the term "physiological acceptable salts" is generally meant to encompass those salts, as well as aqueous solutions made therefrom, having the chemical and biological properties described above.

The term "excipient" is a term of the pharmaceutical arts that generally refers to a pharmacologically inert substance or composition that serves as a delivery vehicle or carrier medium for a drug or bioactive composition. An excipient may include one or more binders, stabilizers, fillers, lubricants, preservatives and the like. A variety of compositions or agents that serve as excipient are known in the art and include, by way of non-limiting example only, certain polymers, small carbohydrates, amphiphilic molecules. Specific example of agents that may serve as an excipient in the presently disclosed embodiments may include, for example amino acids (e.g. glutamic acid), physiological acceptable salts, sodium phosphates, and small polypeptides. Nevertheless, it will readily be appreciated by an ordinary practitioner of the art that various other excipients may be employed during the practice of the invention without departing from the spirit and scope thereof.

As used herein, the term "an osteoinductive composition" generally refers to a composition that induces and/or supports the formation, development and growth of new bone, and/or the remodeling of existing bone. An osteoinductive composition typically includes one or more osteogenic agents. An "osteogenic agent," as used herein, is an agent that can elicit, facilitate and/or maintain the formation and growth of bone tissue. Many osteogenic agents function, at least in part, by stimulating or otherwise regulating the activity of osteoblasts and/or osteoclasts. Exemplary osteogenic agents include certain polypeptide growth factors, such as, osteogenin, Insulin-like Growth Factor (IGF)-I, IGF-II, TGF-βl, TGF-β2 , TGF-β3 , TGF-β4, TGF-β5, osteoinductive factor (OIF), basic Fibroblast Growth Factor (bFGF), acidic Fibroblast Growth Factor (aFGF), Platelet-Derived Growth Factor (PDGF), vascular endothelial growth factor (VEGF), Growth Hormone (GH), growth and differentiation factors (GDF)-5 through 9, osteogenic protein- 1 (OP-I) and any one of the many known bone morphogenic proteins (BMPs), including but not limited to BMP-I, BMP-2, BMP-2A, BMP-2B, BMP-3, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-8b, BMP-9, BMP-10, BMP-Il, BMP- 12, BMP-13, BMP-14, BMP-15. An osteoinductive composition may include one or more agents that support the formation, development and growth of new bone, and/or the remodeling thereof. Typical examples of compounds that function in such a supportive manner include, though are not limited to, extracellular matrix-associated bone proteins (e.g., alkaline phosphatase, osteocalcin, bone sialoprotein (BSP) and osteocalcin in secreted phosphoprotein (SPP)-I, type I collagen, fibronectin, osteonectin, thrombospondin, matrix-gla-protein, SPARC, alkaline phosphatase and osteopontin). As used herein, the term "growth factor" generally refers to a factor, typically a polypeptide that affects some aspect of the growth and/or differentiation of cells, tissues, organs, or organisms.

As used herein, the term "bone morphogenic protein," or "BMP" generally refers to a group of polypeptide growth factors belonging to the TGF- β superfamily. BMPs are widely expressed in many tissues, though many function, at least in part, by influencing the formation, maintenance, structure or remodeling of bone or other calcified tissues. Members of the BMP family are potentially useful as therapeutics. For example, BMP-2 has been shown in clinical studies to be of use in the treatment of a variety of bone-related conditions. Typically, therapeutic dose ranges for aqueous solution containing BMPs range from about 0.01 to about 50 mg/ml, more typically, from about 0.5 to about 5 mg/ml of the BMP, although other dose ranges are possible depending on the specifics of the case and the desired effect.

As used herein, the term "bone protein" generally refers to a polypeptide factor that supports the growth, remodeling, mineralization or maintenance of calcified tissues. Bone proteins are typically components of, or associate with cells and structures that form extracellular matrix structures. Typical examples of bone proteins may include, though are not limited to, alkaline phosphatase, osteocalcin, bone sialoprotein (BSP) and osteocalcin in secreted phosphoprotein (SPP)-I, type I collagen, type IV collagen, fibronectin, osteonectin, thrombospondin, matrix-gla-protein, SPARC, alkaline phosphatase and osteopontin. One or more bone proteins may be included in an osteoinductive composition. As used herein, the term "antibiotic" generally refers to a naturally occurring, synthetic or semi- synthetic chemical substance that is derivable from a mold or bacterium that, when diluted in an aqueous medium, kills or inhibits the growth of microorganisms and can cure or treat infection. As used herein, the term "analgesic" is used in reference to a pharmacologically active agent or composition that alleviates pain without causing loss of consciousness

As used herein, the term "polypeptide" or "protein" generally refers to a naturally occurring, recombinant or synthetic polymer of amino acids, regardless of length or post- translational modification (e.g., cleavage, phosphorylation, glycosylation, acetylation, methylation, isomerization, reduction, farnesylation, etc...), that are covalently coupled to each other by sequential peptide bonds. Although a "large" polypeptide is typically referred to in the art as a "protein" the terms "polypeptide" and "protein" are often used interchangeably. The term "portion", as used herein in the context of a polypeptide (as in "a portion of a given polypeptide/polynucleotide") generally refers to fragments of that molecule. The fragments may range in size from three amino acid or nucleotide residues to the entire molecule minus one amino acid or nucleotide. Thus, for example, a polypeptide "comprising at least a portion of the polypeptide sequence" encompasses the polypeptide defined by the sequence, and fragments thereof, including but not limited to the entire polypeptide minus one amino acid. A polypeptide may be made using recombinant means, or may be isolated from its natural source (i.e., partially purified).

As used herein, the term "recombinant," when used in reference to a polypeptide, generally refers to a protein, or a fragment thereof, that is made and/or at least partially purified using recombinant DNA technology. Techniques for the production of recombinant proteins are widely known in the art. Briefly, a nucleotide sequence encoding the portion of the protein that is to be expressed is inserted in frame to an expression vector (e.g., a viral, bacterial, fungal, yeast, plant, insect or mammalian expression vector) and said expression vector is introduced into an appropriate cell system in culture. The recombinant protein produced by cultured cells may be at least partially purified using a variety of techniques. General guidance in techniques used for the production of recombinant proteins may be found, for example, in Sambrook, J. et al. Molecular Cloning: A Laboratory Manual. 2^nd, Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, which is incorporated herein by reference. When referring to a recombinant protein, such as e.g., BMP-2, the skilled practitioner will generally use the notation "rBMP-2," where "r" denotes "recombinant." Similarly, when referring to a recombinant protein of a given species (such as, e.g., humans), the notation "rh" will be used. Thus, a preparation of a recombinant human BMP protein may be referred to as "rhBMP."

As used herein, the term "at least partially purified" when used in the context of a polypeptide or a composition containing a polypeptide, generally refers to an expressed polypeptide that is substantially free of other cellular material, or culture medium that was present during the production thereof. General Description CALCIUM PHOSPHATE CEMENTS

Calcium phosphate cements suitable for use with the presently described embodiments, including their method of manufacture and use, may include without limitation, those disclosed in U.S. Patent Nos. 6,379,453 and 6,840,995 to Lin et al., entitled "PROCESS FOR PRODUCING FAST SETTING, BIORESORBABLE CALCIUM PHOSPHATE CEMENT"; U.S. Patent Appl. Publ. No. 2004/0031420 by Lin et al., entitled "CALCIUM PHOSPHATE CEMENT, USE AND PREPARATION THEREOF"; U.S. Patent No. 6,960,249 to Lin et al. entitled "TETRACALCIUM PHOSPHATE (TTCP) HAVING CALCIUM PHOSPHATE WHISKER ON SURFACE"; U.S. PATENT APPL. PUBL. NO. 2004-0175320, by Lin et al. entitled "TETRACALCIUM PHOSPHATE (TTCP) HAVING CALCIUM PHOSPHATE WHISKER ON SURFACE AND PROCESS FOR PREPARING THE SAME"; Int'l Patent Appl. Publ. No. WO 2004/094335 by Lin et al entitled "CALCIUM PHOSPHATE CEMENT, USE AND PREPARATION THEREOF"; U.S. Patent Appl. Publ. No. 2005/0076813 by Lin et al. entitled "PROCESS FOR PRODUCING FAST-SETTING BIORESORBABLE CALCIUM PHOSPHATE CEMENT"; U.S. Patent Appl. Publ. No. 2005-0069479 by Lin et al. entitled "METHOD OF INCREASING WORKING TIME OF TETRACALCIUM PHOSPHATE CEMENT PASTE"; U.S. Patent Appl. Publ. No. 2005-0271741by Lin et al. entitled "CALCIUM PHOSPHATE CEMENT, USE AND PREPARATION THEREOF"; U.S. Patent Appl. Publ. No. 2005-0271740 by Lin et al. entitled "CALCIUM PHOSPHATE CEMENT, USE AND PREPARATION THEREOF"; U.S. Patent Appl. Publ. No. 2005-0271742 by Lin et al. entitled "CALCIUM PHOSPHATE CEMENT, USE AND PREPARATION THEREOF"; U.S. Patent Appl. Publ. No. 2005-0268819 by Lin et al. entitled "CALCIUM PHOSPHATE CEMENT, USE AND PREPARATION THEREOF"; U.S. Patent Appl. Publ. No. 2005- 0279252 by Lin et al. entitled "TETRACALCIUM PHOSPHATE (TTCP) HAVING CALCIUM PHOSPHATE WHISKER ON SURFACE"; U.S. Patent Appl. Publ. No. 2005-0268820 by Lin et al. entitled "TETRACALCIUM PHOSPHATE (TTCP) HAVING CALCIUM PHOSPHATE WHISKER ON SURFACE"; U.S. Patent Appl. Publ. No. 2005-0268821 by Lin et al. entitled "TETRACALCIUM PHOSPHATE (TTCP) HAVING CALCIUM PHOSPHATE WHISKER ON SURFACE"; U.S. Patent Appl. Publ. No. 2005-0274287 by Lin et al. entitled "TETRACALCIUM PHOSPHATE (TTCP) HAVING CALCIUM PHOSPHATE WHISKER ON SURFACE AND PROCESS FOR PREPARING THE SAME"; U.S. Patent Appl. Publ. No. 2005-0274286 by Lin et al. entitled "TETRACALCIUM PHOSPHATE (TTCP) HAVING CALCIUM PHOSPHATE WHISKER ON SURFACE AND PROCESS FOR PREPARING THE SAME"; U.S. Patent Appl. Publ. No. 2005-0274282 by Lin et al. entitled

"TETRACALCIUM PHOSPHATE (TTCP) HAVING CALCIUM PHOSPHATE WHISKER ON SURFACE AND PROCESS FOR PREPARING THE SAME"; U.S. Patent Appl. Publ. No. 2005-0274288 by Lin et al. entitled "PROCESS FOR PRODUCING FAST-SETTING BIORESORBABLE CALCIUM PHOSPHATE CEMENT"; U.S. Patent Appl. Publ. No. 2005- 0279256 by Lin et al. entitled "METHOD OF INCREASING WORKING TIME OF

TETRACALCIUM PHOSPHATE CEMENT PASTE"; U.S. Patent Appl. Publ. No. 2006- 0011100 by Lin et al. entitled "PROCESS FOR PRODUCING FAST-SETTING BIORESORBABLE CALCIUM PHOSPHATE CEMENT"; and U.S. Patent Appl. Publ. No. 2006-0011099 by Lin et al. entitled "PROCESS FOR PRODUCING FAST-SETTING BIORESORBABLE CALCIUM PHOSPHATE CEMENT" all of which are commonly owned with the present application and the entire contents of which are hereby incorporated by reference in their entirety as though fully set forth herein. The cements are equally suitable for the production of injectable biomaterials (e.g., injectable CPC formulations), or for the production of hardened CPC implants. In some embodiments, calcium phosphate cements may be formed from acidic calcium phosphates (e.g., calcium phosphates having a calcium to phosphorous ratio of less than 1.33), basic calcium phosphates (e.g., calcium phosphates having a calcium to phosphorous ratio of greater than 1.33) or combinations of acidic and basic calcium phosphates. The presently described CPCs may optionally include one or more bioactive compositions dispersed or dissolved therein, such as are described in detail below.

Particularly suited to the presently described embodiments are CPCs made using calcium phosphate particles having whiskers on the surface of the particles, such as are disclosed in the above-cited references and incorporate by reference herein. Without being bound by any particular mechanism of action, it is believed that the whiskers described in these references increase the surface area of cement particles and allow for improved cementing reactions to occur, resulting in hardened materials having improved compressive strength. Additionally, and by virtue of their ability to form interlocking complexes with the whiskers of adjacent particles, surface whiskers advantageously allow a CPC paste to be non-dispersive in aqueous solutions. Thus, these non-dispersive pastes are well suited to therapeutic applications in which a CPC paste is injected to a site the body of the subject where there exists the possibility that the paste would be washed away by body fluids prior to the hardening thereof. Methods for preparing such compositions are disclosed in the above-identified patent references. An additional method will now be disclosed.

In one embodiment, TTCP particles having an average diameter of <500 μm and are obtained. Such particles may be prepared according to any procedure known in the art, including but not limited to the procedures set forth in the above identified patent references. One exemplary process for preparing TTCP particles for use in preparing the compositions of the present application may include combining Dibasic Calcium Phosphate, Anhydrate (a.k.a.

DCPA; CaHPO₄) (or alternatively Calcium pyrophosphate (Ca₂P₂O₇)) with calcium carbonate (CaCO₃) such that the Ca/P molar ratio is >2.0. For example, 1008.73 grams of dibasic calcium phosphate, anhydrate may be combined with 816.270 grams of calcium carbonate such that the Ca/P molar ratio is 2.1. The combined powders are blended in alcohol (IVS=O.6ml/gm), and the excess alcohol is removed, such as by vacuum filtration and/or evaporation in a drying oven. The dried powder is lightly broken up, such as in a bowl with a spatula or pestle, and fired in a furnace. The typical firing profile when calcium pyrophosphate is used is immediate ramping to 100⁰C at 20°C/minute with a 0 to 4 hour dwell time, followed by a temperature ramp at 5°C/minute up to 800⁰C; followed by ramping at 10°C/minute up to 1200⁰C; followed by ramping at 4°C/minute up to 1400⁰C with final dwell time of 12 hours. Alternatively, when DCPA is used, in order to accommodate the loss of hydrogen and oxygen as water at lower temperatures, the filled crucibles are fired in a furnace with a temperature profile of: heating up to 100⁰C immediately at 20°C/minute with a dwell time of 0 to 4 hours; increasing the temperature 5°C/minute up to 600⁰C; increasing the temperature at 10°C/minute up to 1200⁰C; increasing the temperature at 4°C/minute up to 1400⁰C with dwell time of 12 hours. In either case, after the dwell time is complete, the furnace is allowed to cool to 1000⁰C at the natural cooling rate of the furnace (~10°/min). When the temperature drops below about 1000⁰C the furnace door is opened to speed cooling to room temperature.

The cooled tetracalcium phosphate cakes are crushed to <500microns then milled to a bimodal distribution where 50% of the particles are below approximately 7 to 11 microns. Typical final milling can be performed using a ball mill at 60 rpm in approximately 45 to 60 minutes.

DCPA may be processed for use in a cement composition according to the following procedure. DCPA powder is milled with -40 ml alcohol per 100 grams of DCPA until 50% of the particles are below about 2.5 microns. Typical milling time required at 60 rpm is 3 hours. The alcohol is then removed from the DCPA by drying and the mill media is then removed by sieving. The milled TTCP and processed DCPA are combined in molar quantities between 1:1 to

1:2 and homogenized. The homogenate is subjected to a first whiskering process using a first whiskering solution. An exemplary first whiskering solution may be DI water chilled to 0°- 15⁰C. The first whiskering step is performed at liquid/solid of 22-44ml of first whiskering solution for every gram of combined powders to be whiskered. The powder and liquid are combined with stirring for several minutes (e.g. ~5min). The powder is separated from the solution, for example by vacuum filtration. The captured powder is then rinsed 1 to 3 times with chilled rinse solutions. In certain cases the rinse solutions may contain 0 to 1 OmMoI MgCl₂. Typically the final rinse is performed with DI water without MgCl₂. An amount of 14.67ml of rinse solution is used for every gram of combined starting powders. The excess water is dried off in a drying oven at 5O⁰C to HO⁰C.

A second whiskering solution is prepared using 1 part ortho-phosphoric acid with 58.65 parts DI water. The combined powders already whiskered once are whiskered a second time using the second whiskering at liquid/solid of 0.32 ml per gram powders and dried in an oven at 50°C to HO⁰C. The whiskered cement powders are dry milled for approximately 2 to 60 minutes using a mortar and pestle or a ball mill to achieve a particle size distribution such that 50% of the particles are below approximately 3.5 to 6.5 microns and more preferably below 4.4 to 5.2 microns. A portion of the dry milled powder is then milled further such that 50% of the particles are below approximately 3.5 microns and the specific surface area is greater than about 4m²/g. This can be accomplished in a mechanical mill such as a ball mill using alcohol in the ratio of 0.4ml alcohol per gram of powder. Typical milling time at 60 rpm is 3.5 hours. A mixture of the two different particle sizes is then blended with calcium oxide in the amount of 0.5% to 1.0% to form the final cement powder mixture. The typical mixture of dry milled and wet milled powders is 15% to 100% dry milled powder by weight. One embodiment uses a mixture that is 30% dry milled and 70% wet milled powders.

The final powder is mixed with the setting solution using a spatula or equivalent mixing device at a liquid/solid of about 0.27-0.53 (depending on the desired consistency).

In an embodiment, whiskered TTCP particles may be contacted with a setting solution and heated to result in an hardened apatitic cement suitable for use as an injectable bone filler material, or for use in the manufacture of prosthetic bone implants.

Modified calcium phosphate cement compositions suited for use in the presently described embodiments may be chosen according certain chemical and/or physical properties that are advantageous for therapeutic use. It is desirable that the constituent CPCs used herein have the ability to harden into cements having high compressive strength. Typically, a CPC composition will be chosen such that a hardened cement made therefrom has a compressive strength of >30 MP, >50 MPa, or >100 MPa. A CPC composition may also be chosen such that, when mixed with an appropriate setting solution, a paste having sufficient viscosity so as to allow the paste to be injected through a syringe or other aperture to a site within a body or a mold will be formed. The preceding two parameters are, at least in part, related to the density of whiskers on the surface of constituent calcium phosphate particles, and to the density of particles comprising the paste. The density of surface whiskers will typically be in a range such that the resulting material has the desired characteristics of being non-dispersive and able to withstand high compressive forces, while allowing the paste to remain injectable. Typically, such characteristics may be realized when the density of surface whiskers is > 2.0/μm and less than 100/μm².

In order for CPC materials to be of therapeutic use in a point-of-care setting, a paste made therefrom should have a setting time and working time that is greater than 1 minute and less than 45 minutes. U.S. Patent Application No. 2005/0069479 to Lin et al. entitled

"METHOD OF INCREASING WORKING TIME OF TETRACALCIUM PHOSPHATE CEMENT PASTE," discloses methods to manipulate the setting and working times of various calcium phosphate compositions. By heating a TTCP paste to between about 5O⁰C to 35O⁰C for at least one minute, a paste having a working time and setting time of between about 8 to 45 minutes and about 9.5 minutes to about one hour, respectively, is achieved.

FORMULATION OF BONE SUBSTITUTES AS PHARMACEUTICAL CARRIERS

Described herein is a self-setting cementitious composition suitable for use as a bone substitute material. The self-setting cementitious bone substitute composition promotes in some embodiments, the ingrowth of natural bone into the bone replacement material during bone remodeling. The self-setting cementitious bone substitute composition promotes, in some embodiments, the formation of interconnected macropores within the hardened calcium phosphate matrix of the bone substitute material. The self-setting cementitious bone substitute composition functions in some embodiments, as a pharmaceutical carrier that delivers a therapeutic compound to the bone with a controlled release profile (e.g., multi-modal, exhibiting an initial burst followed by gradual release or slow release profile over longer time). The self- setting cementitious bone substitute composition functions, in some embodiments, as a pharmaceutical carrier and that releases about 2% to about 50% of the initial load of therapeutic compound within 10 days. A variety of strategies may be employed to realize controlled release kinetics of bioactive composition from such a composition. Certain structures of the cement may be capable of being substantially or completely reabsorbed by the host tissue.

A bone substitute material capable of acting as a pharmaceutical carrier may include a mixture containing CPC particles in combination with one or more excipients. A variety of excipients suitable for use in the preparation of the subject bone substitute materials are contemplated. Various non-limiting examples of excipients are set forth in Table I.

In certain non-limiting embodiments, an excipient selected for use in preparing the subject bone substitute material may include one or more macropore forming powders. The ratio of CPC particles to macropore forming powder will generally be in the range of about 4:1 to about 1:1. Macropore forming powders suitable for use in accordance with the present disclosure will typically be biocompatible, resorbable, promote the formation of macropores in the calcium phosphate matrix of the hardened bone substitute material, and function as an excipient by promoting the gradual release of a therapeutic compound from the calcium phosphate matrix of the hardened bone substitute material. Exemplary powders having such characteristics include, though are not limited to, synthetic polymers, salts, sugars, sugar alcohols, amino acids and/or oligo- or polypeptides.

Any physiologically acceptable salt may be used in the present embodiments without limitation. Those salts contemplated to be especially suitable for use as porogens according to the presently described embodiments may include, though are not limited to, sodium, potassium and phosphate salts (e.g., NaCl, KCl and sodium phosphates). Likewise, any physiologically acceptable sugar or sugar alcohol may be used as a porogen in the present embodiments without limitation. Particularly contemplated sugars or sugar alcohols suitable for use as porogens according to the presently described embodiments include, though are not limited to, particulate xylitol, mannitol, maltodextrin, or sorbitol, cellulose, cellulose derivatives, polyvinyl pyrrolidone, starch, sucrose, zein, sortitol, glucose, lactose, polysaccharides, polyethylene glycol, sodium starch glycolate, sodium carboxymethyl cellulosemethycellulose. In some embodiments, a preferred porogen may include mannitol crystals having an average size distribution in the range of about 350 μm up to about 2 mm. Acceptable amino acids may include lysine, glycine, glutamate, alanine, or polymers thereof, either alone or in combination with other compounds. Biodegradable polymers suitable for applications described herein may include, though are not limited to, natural or synthetic polypeptides, polylactic acid, chitosan, polylactic acid- polyethylene glycol block copolymer and their derivatives. In some embodiments, suitable biodegradable polymers may include polyesters, poly(L-lactic acid), poly(D,L-lactic acid), poly(glycolic acid), polycaprolactone, block copolymers and copolymers thereof. In an embodiment, at least a portion of the porogen may be formulated as microspheres. In an embodiment, at least a portion of the porogen may be formulated as microfibers. The macropore forming powder may include certain biodegradable polymers, the resorption of which results in an acidic local micro-environment that encourages localized dissolution of hardened CPC matrix, thereby further promoting the release of the therapeutic composition from the calcium phosphate matrix.

In some embodiments, an excipient may be dispersed in a liquid phase (e.g., in the setting liquid), prior to being mixed with the CPC particles to produce the self-setting bone substitute material. See, e.g., Table I.

To form the bone substitute material of the present invention, a mixture containing the CPC particles and the macropore forming powder is mixed with an appropriate volume of a setting liquid to form a self setting, injectable cementitious bone substitute material. Alternatively, the bone substitute material may be prepared by first preparing a dispersion of the excipient in an aqueous phase, and mixing the aqueous phase with the CPC particles to form a self-setting cementitious composition. The density of the injectable cementitious bone substitute material will typically be in the range of about 0.1 g/ml to about 20 g/ml. Alternatively, the Liquid: Powder ratio of the injectable bone substitute material will be in the range of about 0.27 to about 0.53. Setting liquids contemplated for use in the preparation of the subject bone substitute material may include any physiologically acceptable aqueous liquid capable of promoting cementing reactions between individual CPC particles in the powder phase. Exemplary setting liquids suitable for use in the presently disclosed embodiments include, though are not limited to, acidic solutions, a basic solutions, solutions having substantially physiological pH (e.g., 6.0- 8.5), including autologous tissues, such as blood or marrow and their derivatives, or substantially pure water.

Suitable acidic solutions may include solutions containing nitric acid (HNO₃), hydrochloric acid (HCl), phosphoric acid (H₃PO₄), carbonic acid (H₂CO₃), sodium dihydrogen phosphate (NaH₂PO₄), sodium dihydrogen phosphate monohydrate (NaH₂PO4»H₂O), sodium dihydrogen phosphate dihydrate, sodium dihydrogen phosphate dehydrate, potassium dihydrogen phosphate (KH₂PO₄), ammonium dihydrogen phosphate (NH₄H₂PO₄), malic acid, acetic acid, lactic acid, citric acid, malonic acid, succinic acid, glutaric acid, tartaric acid, oxalic acid and their mixture.

Suitable basic solutions may include solutions containing ammonia, ammonium hydroxide, alkali metal hydroxide, alkali earth hydroxide, disodium hydrogen phosphate (Na₂HPO₄), disodium hydrogen phosphate dodecahydrate, disodium hydrogen phosphate heptahydrate, sodium phosphate dodecahydrate (Na₃PO₄* 12H₂O), dipotassium hydrogen phosphate (K₂HPO₄), potassium hydrogen phosphate trihydrate (K₂HPO₄*3H₂O), potassium phosphate tribasic (K₃PO₄), diammonium hydrogen phosphate ((NH₄)₂HPO₄), ammonium phosphate trihydrate ((NH₄)₃PO₄*3H₂O), sodium hydrogen carbonate (NaHCO₃), sodium carbonate Na₂CO₃, and their mixture.

Non-limiting examples of solutions having substantially physiological pH (i.e., a pH in the range of about 6.5 to about 8.5) include solutions containing phosphate ions such as, e.g., a phosphate buffer, phosphate buffered saline (PBS), Hank's solutions, serum or complex solutions of biomolecules (e.g., a mixture of various serum and/or marrow protein in a physiologically buffered aqueous medium).

In one embodiment, a setting liquid may be an aqueous solution suitable for reconstituting a desiccated or otherwise preserved protein or polypeptide mixture. For example, certain embodiments of the present invention are directed to cementitious compositions that include one or more recombinant polypeptide growth factors dispersed therein. In such cases, a setting liquid having a composition suitable for reconstituting recombinant growth factor may be utilized. By way of non-limiting example, in embodiments in which one or more BMP proteins are utilized, the solution in which the recombinant protein preparation is to be reconstituted may itself be used as a setting liquid. In the case of BMPs, such a solution may generally include a buffered aqueous preparation having a pH in the range of about 3.5 to about 5.5, or in the range of about 4.0 to about 7.0, and containing from about 0.05 to about 5 wt.% sucrose, from about 0.5 to about 50 mM glutamate, from about 0.001 vol.% to about 0.1 vol.% polysorbate-80, from about 0.25 wt.% to about 25 wt.% glycine, and from about 0.5 to about 50 mM NaCl. Of course, it will be readily understood by the skilled practitioner that one or more of the above mentioned components may be absent from a final preparation, or that one or more components may be substituted for alternative components, without departing from the sprit and scope of the present disclosure. Likewise, it will be understood that a multitude of other reconstituting buffers may be substituted for that set forth above. Thus, the above is not intended to limit the types of protein reconstitution buffers that may be suitable for use in the presently described embodiments, but rather, the intent is to demonstrate to the skilled practitioner a representative buffer that is suitable for reconstituting a recombinant protein as well as use herein as a suitable setting buffer. Likewise, it will be understood by the skilled practitioner that the presence or absence of a recombinant protein in a setting solution will have no appreciable effect on the suitability of a given reconstitution buffer as a setting liquid (i.e., a buffer having a recombinant protein dissolved therein will generally have the same or similar properties when used as a setting solution as the identical buffer lacking the recombinant protein.

A variety of macropore forming powders that function as excipients and that are suitable for use in the presently described embodiments is set forth in Table II. It will be understood by the skilled practitioner that alternative formulations may be used.

In some embodiments, biodegradable polymers may be mixed with cement powder or setting solution and act as porogen. Without being bound by any particular theory or mechanism of action, it is believed that the degradation of such polymers in situ results in an at least partially acidic micro-environment (e.g., pH < about 7). Acidic conditions in the vicinity of the degrading porogen may enhance localized dissolution of the calcium phosphate mineral matrix, resulting in a porous bioceramic matrix that releases the growth factors from the cement over the time. Polymers such as polylactic acid, polylactic acid-polyethylene glycol block copolymer and their derivatives are used to fabricate microspheres or microfibers and combined with calcium phosphate cement to deliver growth factors. Optionally, the biodegradable polymers may be provided to the subject bone replacement material in combination with one or more additional excipients, such as, for example, molecules that decrease the binding affinity of apatite for peptide growth factors (e.g., certain amino acids such as alanine).

In an embodiment, a therapeutic composition (e.g., a growth factor other drug) may be added to the CPC particles and the macropore forming powder prior to the mixing thereof with the setting liquid. Alternatively, a therapeutic composition may first be dissolved or dispersed in the aqueous phase of the setting liquid prior to the mixture thereof with the CPC particles and the macropore forming powder.

The presently disclosed bone substitute materials are formulated for use as a pharmaceutical carrier for one or more therapeutic compositions. A therapeutic composition may be formulated to include osteoinductive agents, growth factors, antibiotics, analgesics, or various combinations thereof. In an embodiment, a therapeutic composition may include at least one growth factor from the TGF- β superfamily of growth factors, or at least one growth factor from the BMP family of growth factors, or at least one growth factor from the GDF family of growth factors, or at least one growth factor from the IGF family of growth factors, or any combination thereof. In an embodiment, a therapeutic composition may include BMP-2, BMP-4, BMP- 12, or their combination. In certain embodiments, a therapeutic composition may include at least a portion of one or more polypeptides, including but not limited to at least a portion of a polypeptide growth factor, at least a portion of one or more TGF- β superfamily growth factors, at least a portion of one or more BMP growth factors, or various combinations thereof. In an embodiment, a polypeptide for use in an osteoinductive composition as described herein may be at least partially purified. Source materials for at least partial purification of the polypeptides as described herein may include natural source material (e.g., natural bone, bone marrow, cultured cells), or recombinant material (e.g., protein whose expression is facilitated and or enhanced by way of a suitable viral, bacterial, yeast, insect, plant or mammalian protein expression system including a suitable expression vector). In some embodiments, one or more of the bone growth factors may be derived from autogenic bone, allogenic bone, xenogenic bone, or from recombinant sources.

Therapeutic compositions particularly suited for inclusion in the subject bone substitute materials may include one or more bone morphogenetic proteins e.g. BMP-2, BMP-7, BMP-9, GDF-5, GDF-6, and GDF-7, one or more transforming growth factors (e.g., TGF-beta), one or more IGFs (e.g., IGF-I, and IGF-2), or various portions and/or combinations thereof. In an embodiment, autologous bone marrow (e.g., derived from the subject who will be receiving it) or bone-derived TGF-beta, insulin-like growth factors, platelet-derived growth factor and BMP2, or any of the bioactive agents disclosed herein, may be combined with the injectable bone substitute materials. The determination of a therapeutically effective dose range for individual growth factors is within the skill level of the ordinary practitioner of the art. In the case of polypeptide growth factors, therapeutically effective dose ranges will generally be in the range of about 0.1 to about 10 mg protein per cc of self-setting bone substitute material.

In an embodiment, the injectable calcium phosphate cement bone substitute compositions may harden to form a calcium phosphate material. In embodiments in which porogens were included in the paste, the hardened material may be soaked in a physiologically acceptable aqueous solution to promote the dissolution/degradation of at least a portion of the porogen embedded throughout the hardened matrix, thereby creating a network of pores therein. Alternatively, the hardened material may be implanted into a site in the body (e.g., a tooth or a bone). Over time, body fluids in contact with the hardened calcium phosphate material/porogen composite may penetrate the matrix thereof and allow dissolution/degradation of the porogen, thereby creating a network of pores therein. In an embodiment, the self setting cementitious bone substitute carrier compositions may be prepared as a paste made by mixing a dry phase that includes calcium phosphate cement (CPC) particles in combination with at least macropore forming powder; and contacting said dry phase with a liquid phase (i.e., a setting liquid) to form a self-setting cementitious mixture. In one embodiment, a therapeutic composition may be added to the self-setting cementitious mixture. Alternatively, a therapeutic composition may be included as a component of the dry phase prior to the mixing thereof with the liquid phase. In a further alternate embodiment, the therapeutic composition may be included as a component of the liquid phase prior to addition thereof to the dry phase. In yet a further embodiment, the therapeutic composition may be included as a component of both the solid phase and the liquid phase. The ratio of the solid phase to liquid phase of the composition may be sufficient to form a paste that can readily be injected using syringe to a site on a bone or to a mold in order to make an implantable structure. In an embodiment, the ratio of solid phase to liquid phase may be in the range of about 0.1 g/ml to about 20 g/ml.

In an embodiment, the injectable calcium phosphate cement bone substitute compositions may be delivered to a bone defect or to a mold and form a hardened bioresorbable calcium phosphate material. In an embodiment, certain structures of the hardened CPC may be substantially reabsorbed by or released around the site of its application. In certain embodiments, porogens within the hardened calcium phosphate matrix may gradually be removed from the hardened material, leaving pores and/or an interconnected network of porosity dispersed throughout the matrix. In some embodiments, bioactive compositions incorporated in the hardened calcium phosphate material and/or in the porogens may be gradually released to surrounding tissue.

In some embodiments, any bioactive agent that facilitates or stimulates new bone growth (e.g., an osteogenic agent) may be delivered to a bone or to an implant using a suitable excipient combined with injectable calcium phosphate cement as a carrier. In certain embodiments, bioactive compositions may include osteoinductive compositions such as bone morphogenetic proteins e.g. BMP-2, BMP-7, BMP-9, growth differentiation factors (GDF)-5, GDF-6, and GDF- 7, platelet-derived growth factors (PDGFs), transforming growth factors (e.g., TGF-β), insulin- like growth factors (IGF)-I, and IGF-2, autogenic or allogenic bone or bone marrow, bone- derived TGF-β, IGF, and BMP2. EXAMPLES

The following will serve to illustrate, by way of one or more examples, systems and methods for inhibiting, reducing or otherwise disrupting prolactin signaling in pain neurons according to some embodiments. The examples below are non- limiting and are intended to be merely representative of various aspects and features of certain embodiments. Although methods and materials similar or equivalent to those described herein may be used in the application or testing of the present embodiments, suitable methods and materials are described below. EXAMPLE 1

Tables 1 and 2 set forth various bone substitute carrier formulations. The bone substitute carrier formulations were prepared using four different setting solutions: first, water alone; second, BMP reconstituting buffer composed of 0.5% Sucrose, 5mM Glutamate, 0.01% Polysorbate 80, 2.5% Glycine and 5mM NaCl, pH 4.5; third, a solution containing rhBMP-2 (-1.5 mg/ml and ~4 mg/ml) in BMP reconstituting buffer; and fourth, a complex mixture of marrow proteins. The following methods or sources were used to obtain the final porogen compositions.

Polylysine: 0.1% polylysine was added to setting solution (0.7 M phosphate solution, pH 5.6) at the volume ratio of 3:4. Glutamate: Potassium Glutamate monohydrate granules from the bottle were used directly as porogens. The particle size is about 100-400 μm.

Glycine: Glycine granules from the bottle were used directly as porogens. The particle size is about 100-500 μm.

Alanine: Alanine powder from the bottle was used directly as porogens. The particle size is about 50-200 μm.

Polylactic Acid: 1 g of Polylactic acid was dissolved into 5 mL acetonitrile. 5mL of DI water was added to the PLA solution and freeze to -70⁰C and lyophilized. The lyophilized PLA powder was mixed with cement powder at the ratio of 1:3.

Polyethylene Glycol: Polyethylene glycol (MW 2000) was dissolved in 0.7M phosphate setting solution (pH 5.6) at the concentration of 0.5 g/mL.

NaCl/KCl: NaCl and KCl from the bottle was sieved and 50-355 μm particles were used at the NaCl/KCl ratio of 1.

Polyglycolic Acid: Polyglycolic acid granules were used directly. The particle size is about 0.5-3 mm.

Small Mannitol Crystals were prepared using a re-crystallization procedure. The dried mannitol was then ground and sieved through openings of 510 μm (top sieve) and 355 μm (bottom sieve). Very Large Mannitol Crystals (1-4 mm): Large mannitol crystals were prepared using a re-crystallization procedure. The dry mannitol crystals were sieved through openings of 4mm (top sieve) and 2mm (bottom sieve). The resulted mannitol crystals have a diameter of 1-4 mm and length about 5-10 mm.

Large Mannitol Crystals (0.71- lmm): Mannitol crystals (0.71-lmm in length) were prepared by a re-crystallization procedure. The dried crystals sieved through openings of 1 mm (top sieve) and 710 μm (bottom sieve).

Setting % BMP-2 Cytotoxicity Bone

Excipient/ Non- Pore Size Biological time Cohesiveness released after Cytotoxicity after 3X formation in Porogen Dispersibility (μm) activity (min) 10 days dilution vitro

None +++++ +++++ No L32 0.7 0.12 +++ ++++

H -t~H-H- +++++ 0.11 ^■*^■+++ ++++

» ++++ O83 +++. +++.

+++ +++ -200 3.38 0.3 1.07

K*

B +++++ +++++ No l.i 0.16 0.71

D+F ++ ++ -100 7.74 0.57 0.78

++ ++ No 7.13 0.82 0.99 ++++ ++

G +++ +++ 200-300 19.82 0.04 0.03 ++

G+F ++ 200-300 46.43 N/A N/A N/A

I+G +++ +++ 100-300 6.58 +++++ ++

10 ++ N/A 21.15 N/A N/A N/A N/A

A 10 +++++ +++++ No 2.77 N/A N/A N/A N/A

Table I. Properties of various Bone Cement Formulations for Use as Growth Factor Carriers.

Table II.

EXAMPLE 2

BMP-2 release Profile of CPC carrier/ 'excipient

The characterization of growth factor delivery was based on loading and release studies including functional tests, biochemical analysis, and in vitro biological effect of rhBMP-2 from two sources (R&D systems Minneapolis, MN and Wyeth, Madison, NJ). Figures 1 through 4 show the release profiles of rh-BMP2 from various formulations described in Table I. lOμg of reconstituted rhBMP-2 was mixed with setting solution composed of 0.7M Phosphate, pH 4.0, and the final solution mixed with 0.5g of cement powder thoroughly. The resulted paste was gently shaped into a ball and immediately submerged in media containing DMEM with 5% Fetal Calf Serum and placed at 37°C. The media was collected and replaced at different time points to measure the amount of rhBMP-2 released.

Figure 1 shows that a bone substitute material containing excipient A, rhBMP-2 release was increased from 0.3% to 2.7% at 6 days.

Figure 2 shows the rhBMP-2 release profiles of a bone substitute material containing excipient G, excipient F+G, excipient A+F+G, or excipient A+G. The release of the growth factor after 24 hours was significantly increased (from 0.3% to 46%) after adding excipient F+G into the bone substitute formulation.

Figure 3 shows the rhBMP-2 release profiles of a bone substitute material containing excipient H, excipient E, or excipient K. Figure 4 shows the rhBMP-2 release profiles of a bone substitute material containing excipient J, excipients I+G, excipient D, excipient C, or excipient B.

Therefore, by changing the composition of the self-setting bone substitute material, the release profile of rhBMP-2 from said material can be changed.

Figures 5 shows the release profiles of self-setting bone substitute formulation containing excipients H+L. As mentioned earlier this formulation sets with water, with various physiological solutions (e.g., PBS, Hanks, or saline) with the addition of rhBMP-2 (~ 1.5 to 4 mg/mL) reconstituted in buffer solution, or with rhBMP-2 buffer alone. In Figure 5, 11.7% of the total amount of rhBMP-2 loaded (0.25mg) was released from the 0.5g bone substitute composition after 15d. In contrast, when rhBMP-2 was loaded in a much larger volume of self- setting bone substitute material (15g), only 2.29% of the total amount of protein loaded (4.5mg) was released from the specimens in the same period of time (shown in Figure 6). The loading capacity for a self-setting bone substitute formulation as a carrier for rhBMP2 is limited by two factors: the liquid to solid ratio of the cement, and the solubility of the protein. At the lower protein concentration of 1.5mg/ml the loading capacity is maxed at 0.53mg/cc final cement. However, 3mg/ml solutions of rhBMP-2 can yield a loading maximum of 1.06mg/cc. EXAMPLE 3

Cytotoxicity and in vitro biological activity of rhBMP-2 released from the bone substitute material

The cytotoxicity of the various bone substitute formulations was tested on the mouse fibroblastic cell line L929. Briefly, freshly made bone substitute material containing the indicated excipients was prepared and submerged in α-MEM medium containing 5% fetal calf serum at ratio of 5 ml medium per gram of bone substitute, and samples were incubated at 37 ⁰C with gentle shaking for 24 hrs. The media conditioned by the bone substitute material was used to treat L929 cells, and, 24 hrs later, cell proliferation was measured by a live/dead assay, which measures the metabolic activity of the cells. The cell viability was expressed as absorbance and normalized to the control cells cultured with 5% medium. As shown in Figures 7 and 8, all bone substitute carrier formulations showed certain cytotoxicity, which mainly results from increase in osmotically active molecules during the setting of the cement. The cytotoxicity was significantly decreased when the cells were treated with diluted extracting medium (see Figure 9).

Figure 8 shows the cytotoxicity of rhBMP-2 loaded bone substitute material. The preparations were set with rhBMP-2 reconstituted in deionized water and contained a final composition of 0.5% Sucrose, 5mM Glutamate, 0.01% Polysorbate 80, 2.5% Glycine and 5mM NaCl, pH 4.5. The bone substitute was extracted, as explained above, and after 24 and 48 hrs the media was removed and refreshed. The cement-extracting medium showed cytotoxicity after 24 hours only. No cytotoxicity was observed at the 48 hr time point. EXAMPLE 4 RhBMP-2 released from the bone substitute material is bioactive To evaluate the biological activity of BMP-2 released from the bone substitute composition, the released growth factor was applied to the mouse pre-myocyte cell line C2C12, which respond to BMP-2 by a marked induction in alkaline phosphatase (ALP) activity, indicating the progression towards an osteoblastic phenotype. A self-setting bone substitute composition containing excipients H+L was mixed with rhBMP-2 reconstituted solution into paste and then submerged in 5% DMEM medium. The conditioned medium was collected at different time point and used to treat C2C12 cells for 5 days. Figure 9 shows the induction of alkaline phosphatase activity on C2C12 cells.

The BMP-2 conditioned medium showed significantly induction of ALP activity on C2C12 cells, which indicates that the released BMP-2 from the bone substitute composition is biologically active. The bone inductive effect of the composition with rhBMP2 was evaluated in vitro by measuring collagen production and subsequent extracellular matrix mineralization. The mouse pre-osteoblastic cell line MC3T3-E1 (subclone 4) was used for these assays and cells were grown directly on the Putty and on tissue culture plastic and treated with media conditioned with bone substitute composition (H+L)/rhBMP2 constructs. The bone substitute compositions plus rhBMP-2 samples were freshly made and submerged in MC3T3-E1 medium containing 10% serum. After 2 hours of equilibration and setting the cured cement was removed and MC3T3-E1 cells were directly seeded on the cement samples. The tissue culture media was changed to 5% serum medium after 24 hrs of culture and for the duration of the treatment. SEM images of MC3T3-E1 cells cultured on cement after 3 weeks are shown in Figure 10. Interwoven collagen matrix and new hydroxyapatite deposition (arrows) were observed on the bone substitute material. Cells growing on plastic surrounding the implant also showed strong calcification in the samples containing the growth factor. Newly formed hydroxyapatite stained bright red in the presence of Alizarin Red Stain (Figure 10). In short, the bone substitute composition effectively delivered rhBMP2 and the construct elicited a strong osteogenic response by direct stimulation of the cells attached to it and those in the immediate environment. EXAMPLE 5

Structure and Mechanical Strength of Osteofix carriers Figure 11 shows the porous structure of Osteofix carrier containing excipients H (Larger

Pores) and I (Smaller Pores). The channel like macropores of the hardened bone substitute composition makes it fully permeable thus facilitating bone ingrowth. The average porosity of this bone substitute carrier is 65.12+0.40%. EXAMPLE 6 Mechanical strength of hardened material

The mechanical strength of the bone substitute formulation with excipient H is shown in Figure 12. After 24 hours of curing, the cement reached its final compressive strength of 4.18 +0.81 MPa. The compressive strength between 1 and 24 hours decreases as pore formation and interconnectivity increases. In this patent, certain U.S. patents, U.S. patent applications, and other materials (e.g., articles) have been incorporated by reference. The text of such U.S. patents, U.S. patent applications, and other materials is, however, only incorporated by reference to the extent that no conflict exists between such text and the other statements and drawings set forth herein. In the event of such conflict, then any such conflicting text in such incorporated by reference U.S. patents, U.S. patent applications, and other materials is specifically not incorporated by reference in this patent.

Further modifications and alternative embodiments of various aspects of the invention may be apparent to those skilled in the art in view of this description. Accordingly, this description is to be construed as illustrative only and is for the purpose of teaching those skilled in the art the general manner of carrying out the invention. It is to be understood that the forms of the invention shown and described herein are to be taken as the presently preferred embodiments. Elements and materials may be substituted for those illustrated and described herein, parts and processes may be reversed, and certain features of the invention may be utilized independently, all as would be apparent to one skilled in the art after having the benefit of this description to the invention. Changes may be made in the elements described herein without departing from the spirit and scope of the invention as described in the following claims. In addition, it is to be understood that features described herein independently may, in certain embodiments, be combined.

Claims

WHAT IS CLAIMED IS:

1. A bone substitute composition comprising: calcium phosphate cement particles; a setting solution; and at least one polypeptide growth factor.

2. The bone substitute composition of claim 1, further comprising at least one excipient.

3. The bone substitute composition of claim 2, wherein the excipient is selected such that the bone substitute composition releases more of the polypeptide growth factor into an aqueous medium than an identical bone substitute composition which lacks the excipient.

4. The bone substitute composition of claim 2, wherein the excipient is selected such that at least about 4% of the growth factor is released from the bone substitute composition when immersed in an aqueous medium for 10 days.

5. The bone substitute composition of claim 2, wherein the excipient is selected such that between about 4% to about 46% of the growth factor is released from the bone substitute composition when immersed in an aqueous medium for 10 days.

6. The bone substitute composition of claim 2, wherein the excipient comprises a macropore forming powder.

7. The bone substitute composition of claim 6, wherein the particles of the macropore forming powder have an average diameter in the range of about 100 μm to about 5000 μm and length of lOOμm to 30 mm.

8. The bone substitute composition of claim 6, wherein the ratio of macropore forming powder: calcium phosphate cement particles is in the range of about 1:4 to about 1:1.

9. The bone substitute composition of claim 6, wherein the ratio of macropore forming powder: calcium phosphate cement particles is in the range of about 0.05 to about 2.

10. The bone substitute composition of claim 6, wherein the ratio of setting solution: dry components is in the range of about 0.1 to about 0.55.

11. The bone substitute composition of claim 6, wherein the macropore forming powder comprises at least one of a salt, a sugar, a sugar alcohol, an amino acid, a biodegradable polymer, or their combination.

12. The bone substitute composition of claim 6, wherein the macropore forming powder comprises one or more powders selected from the list consisting of polylysine, glutamate, glycine, alanine, polyethylene glycol, polylactic acid, NaCl, KCl, large mannitol crystals (about 0.5 mm to about 30 mm in length, 0.5 mm to 5 mm in diameter), small mannitol crystals (about 0.1 mm to 2 mm in length, 0.1 mm to 0.5 mm in diameter), polyglycolic acid, polyethylene oxide-c-polylactic acid (PLA-co- PEO), and NaH₂PO₄, or combinations thereof.

13. The bone substitute composition of claim 6, wherein the macropore forming powder comprises large mannitol crystals, alanine, or a mixture of large mannitol crystals and NaH₂PO₄.

14. The bone substitute composition of claim 6, wherein the macropore forming powder comprises a mixture of large mannitol crystals and NaH₂PO₄.

15. The bone substitute composition of claiml4, wherein the ratio of large mannitol crystals to NaH₂PO₄ is about 2: about 0.15.

16. The bone substitute composition of claiml4, wherein the ratio of large mannitol crystals to cement powder is 0.05 to 2.

17. The bone substitute composition of claiml4, wherein the ratio of cement particles/large mannitol/NaH₂PO₄ is about 3/about 2/about 0.15.

18. The bone substitute composition of claim 1, wherein the polypeptide growth factor is BMP-2, rhBMP-2, or a mixture thereof.

19. The bone substitute composition of claim 1, wherein the aqueous setting solution is an acidic solution, a basic solution, pure water, or BMP-2 reconstitution buffer.

20. A bone substitute composition comprising: a self-hardening cementitious composition comprising a calcium phosphate matrix, at least one macropore forming powder, wherein at least a portion of the particles of the macropore forming powder are dispersed throughout said matrix; and at least one polypeptide growth factor; wherein the macropore forming powder is capable of functioning as an excipient and is selected such that the bone substitute composition releases more of the polypeptide growth factor into an aqueous medium than an identical bone substitute composition which lacks the macropore forming powder.

21. The bone substitute composition of claim 20, wherein said matrix, when hardened, is substantially comprised of hydroxyapatite.

22. A bone substitute composition comprising: a self-hardening cementitious composition comprising a calcium phosphate matrix, at least one macropore forming powder, wherein at least a portion of the particles of the macropore forming powder are dispersed throughout said matrix; and at least one polypeptide growth factor; wherein the macropore forming powder is capable of functioning as an excipient and is selected such that the bone substitute composition, when immersed in an aqueous medium for about 10 days, releases about 4 to about 35 fold more of the polypeptide growth factor into the aqueous medium than an identical bone substitute composition which lacks the macropore forming powder.

23. A bone substitute composition comprising: a self-hardening cementitious composition comprising a calcium phosphate matrix, at least one macropore forming powder, wherein at least a portion of the particles of the macropore forming powder are dispersed throughout said matrix; and at least one polypeptide growth factor; wherein at least about 4% of the growth factor is released from the bone substitute composition when immersed in an aqueous medium for 10 days.

24. The bone substitute composition of claim 23, wherein at least about 11% of the growth factor is released from the bone substitute composition when immersed in an aqueous solution for 10 days.

25. The bone substitute composition of claim 23, wherein at least about 46% of the growth factor is released from the bone substitute composition when immersed in an aqueous solution for 10 days.

26. A bone substitute composition comprising: calcium phosphate cement particles; a setting solution; and at least one bone growth factor.