Government Sponsorship

This invention was made with Government support under Grant No. DE 09421 awarded by the NIH. The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention is directed to a dry particulate agglomerate composition capable of producing phase-pure hydroxyapatite without additional sources of calcium and/or phosphate in the presence of an aqueous liquid consisting essentially of agglomerates, each individual agglomerate being a homogenous mixture of (i)

CaHP0 ₄ *2H ₂ 0 particles and (ii) particles of a calcium source comprising Ca ₄ (P0 ₄ ) ₂ 0 wherein said CaHP0 ₄ *2H ₂ 0 and said calcium source comprising Ca ₄ (P0) ₂ 0 are present in an amount sufficient to form phase-pure HAp having a Ca/P ratio of 1.5 to 1.67 and a method for its production. The dry particulate agglomerate composition

of the present invention is capable of forming phase- pure hydroxyapatite in vivo at physiological temperatures within 1 hour in the presence of an aqueous liquid. The present invention is also directed to making a synthetic bone-like or dental composition comprising said particulate agglomerate composition and optionally, a polymeric material capable of promoting mineralization of hydroxyapatite, which are useful for fixing prosthetic devices, useful as bone substitutes to directly fill bone defects, to provide substrates for cartilage, and to repair teeth, and methods of making such preparations.

Description of Related Art The microstructural features of teeth and bone and the processes by which remodeling occurs are of importance in determining desirable microstructures in bone-like substitutes. Because bone substitutes must emulate the function of bone, bone must bond well with them. The nature of this bonding will depend on the pore sizes in the polymeric material capable of forming mineralization of hydroxyapatite. It has been shown that the response of bone to porous implants depends on the pore size. Bone growth occurs in close apposition to an implant which contain 40 μm pores regardless of whether the implant is a metallic or a ceramic material. For pore sizes of 100 μm, bone will fill in the pore and for pores sizes in excess of approximately 150 μm Haversion systems will develop and facilitate the formation of a strong bond with the bone substitute. The ability to directly control microstructural development in a prosthesis composed of a resorbable material and consequently to control its porosity has a major influence on bone intergrowth and prosthesis remodeling. Unfortunately, mechanical integrity and porosity are conflicting functional requirements. Thus,

it is unlikely that a single material would be capable of meeting the function requirements of bone. Indeed, none has been found. The alternative is to develop a composite which would exhibit adequate mechanical properties along with the porosity needed to meet the biological requirements for remodeling.

There have been many attempts to prepare a substitute bone material and there are numerous patents disclosing methods of preparation of such substitute bone materials. U.S. Patent No. 4,440,750 to Glowacki, et al . discloses a plastic dispersion of demineralized bone powder and reconstituted native atelopeptide collagen fibers in a continuous aqueous phase to repair or construct bone by injecting or implanting it at the repair or constructive site. U.S. Patent No. 4,516,276 to Mittelmeier employs collagen as a fleece described as a grid or network which is than dusted with apatite powder or granules, or the fiber material is mixed with mineral before being formed into layers for implantation into bone. Some patents claim the use of porous hydroxyapatite, for example U.S. Patent No. 4,629,464 discloses a method for preparation of artificial bone material where sintered microporous hydroxyapatite is used either in a granular or slurried form in a physiological saline solution or in a form of a shaped prosthetic bone substitute. The prepared bone substitute is characterized by an open porous structure. Such an open porous structure allows for ingrowth of natural tissue. Another method and composition of material promoting the growth of bone is disclosed in U.S. Patent No. 5,073,114. The bone growing composition includes two sizes of hydroxyapatite for supporting the growth of the bone, tetracycline for its antibiotic effect, freeze-dried decalcified human bone for promoting bone growth and fibronectin for promoting connective tissue generation and for gelling the bone growing composition. U.S. Patent No. 4,776,890

introduces a process for obtaining a matrix of mineral particles in reconstituted atelopeptide collagen comprising reconstituting a mixture of mineral particles with collagen in solution. However, none of the above discussed bone substitutes undergo reaction in vivo which would lead to their exhibiting mechanical properties comparable to those of natural bone.

U.S. Patent Nos. 4,880,610; 5,047,031; and 5,053,212 to Constantz disclose calcium phosphate compositions useful as cements. The process involves merely mixing two dry ingredients, i.e., a calcium source and a phosphoric acid source under reacting conditions to form a kneadable mixture. None of these patents disclose or suggest a dry particulate agglomerate composition, capable of forming phase-pure hydroxyapatite without additional sources of calcium and/or phosphate in the presence of an aqueous liquid, prepared by mixing CaHP0 ₄ *2H ₂ 0 (brushite) particles and particles of a calcium source comprising Ca ₄ (P0 ₄ ) ₂ 0 in a non-aqueous liquid which will not allow the particles to react with each other, and then removing the non-aqueous liquid according to the present invention, and which can be stored in a dry state and which are stable over long periods of time. In U.S. Patent 5,053,212, water is liberated in the mechanical mixing of the calcium source with the acidic phosphate source under reacting conditions. Disadvantageously, this liberation of water is free to combine with the calcium and acidic phosphate sources which results in calcium phosphates which are slow to react to form HAp, and upon reaction to form HAp again release water, thereby increasing the porosity and limiting the mechanical strength of the resultant product. W. Brown and Chow, U.S. Patent 4,612,053, disclose mechanically mixing Ca ₄ (P0 ₄ ) ₂ 0 with at least one other calcium phosphate selected from the group consisting of

CaHPO^H _j O, CaHP0 ₄ , Ca,H ₂ (P0 ₄ ) ₄ .5H _j O (i.e., Ca,(HP0) ₂ (P0 ₄ ) ₄ .5H_0), α-Ca ₃ (P0 ₄ ) ₂ , β-Ca ₃ (P0 ₄ ) ₂ and modified Ca,(P0 ₄ ) ₂ for use in a dental restorative powder, paste and/or slurry. This mechanical mixing does not result in a dry particulate agglomerate composition wherein each agglomerate is a homogenous mixture of Ca ₄ (P0 ₄ ) ₂ 0 and the recited calcium phosphates which would allow phase pure HAp to form within an hour. Moreover, in this patent activation to form HAp occurs by the addition of phosphoric acid which results in a pH below physiologically acceptable levels. Thus, this patent does not disclose or suggest the use of, or the inventive dry particulate agglomerate composition capable of forming hydroxyapatite without additional sources of calcium and/or phosphate, and which said particles can be stored in a dry state for long periods of time and which when mixed with a physiologically acceptable aqueous liquid, can completely react to form phase-pure hydroxyapatite in vivo within a surgically relevant time of less than 1 hour, particularly less than 30 minutes, as in the present invention.

The above discussed patents to W. Brown and Chow and Constanz rely on grinding mixtures of particles to a high fineness, which particles in admixture are then reacted in an aqueous solution to form hydroxyapatite.

Neither U.S. Patent 4,612,053, nor U.S. Patents 4,880,610, 5,047,031 or 5,053,212 disclose the preparation of a dry particulate agglomerate composition wherein each agglomerate is a homogenous mixture of CaHP0 ₄ *2H ₂ 0 particles and particles of a calcium source comprising Ca ₄ (P0 ₄ ) ₂ 0 capable of forming phase pure hydroxyapatite wherein the particulate agglomerates are formed as a result of mixing CaHP0 ₄ *2H ₂ 0 particles and particles of a calcium source comprising Ca ₄ (P0 ₄ ) ₂ 0 to a degree of homogeneity which can only be attained when said mixing occurs in a non-aqueous liquid and utilizing

the same in preparing hydroxyapatite compositions useful as a bone-like substitute or a dental composition as in the present invention.

The present invention is directed to a method of preparation of the particulate agglomerate composition, wherein each particulate agglomerate is an intimate mixture of CaHP0 ₄ *2H ₂ 0 particles and a calcium source comprising Ca ₄ (P0 ₄ ) ₂ 0 which are capable of forming phase- pure hydroxyapatite and which can be stored over a long period of time which provided phase-pure HAp, besides exhibiting biocompatibility and the mechanical characteristics required for teeth or artificial bone, can be formed in a surgically relevant time, e.g., usually under 1 hour, particularly under 30 minutes. Most of the prior art requires prosthesis preparation with cured hydroxyapatite in advance of surgery. This complicates treatment and requires taking measurements and separately fitting of the hydroxyapatite implant. The present invention introduces an novel way of preparing phase pure hydroxyapatite at physiological temperature in a surgically relevant time, i.e., hydroxyapatite formation is complete within 1 hour, usually within 30 minutes.

The advantages of using the dry particulate agglomerate composition of the present invention to make hydroxyapatite as a bone substitute are that the properties can be tailored to serve a variety of needs. These include development of the microstructures of both cancellous (porous) and cortical (dense) bone. In addition, the proportions of the constituent ingredients can be adjusted accordingly as a means of controlling the rate and extent of remodeling. Remodeling is the term used to describe the process of bone replacement. Remodeling in synthetic bone may be particularly desirable when younger individuals are involved. Remodeling can be achieved when defects are filled with osteoinductive (bone-forming) materials.

The formation of hydroxyapatite with the particulate agglomerate composition of the present invention in vivo is important in a variety of circumstances like gap filling and bone fragment st abilization. A synthetic material to fill bone defects that is compatible with decalcified bone has not been before identified and there has been a long felt need in the art. The initiation of any bone remodeling procedure will occur more rapidly if a hydroxyapatite- based prosthesis can be placed into close or microscopic apposition with bone. As a result of the present invention, the present method is amenable to placement of a bone-like composition of the reactants into close apposition with bone. This would eliminate the existence of a macroscopic gap between a synthetic hydroxyapatite-based prosthesis and bone or to fill gaps between a metallic prosthesis and bone. The development of union would be promoted in both instances, as a result of the present invention.

SUMMARY OF THE INVENTION The present invention is directed to a dry particulate agglomerate composition capable of producing phase-pure hydroxyapatite without additional sources of calcium and/or phosphate in the presence of an aqueous liquid consisting essentially of agglomerates, each individual agglomerate being a homogenous mixture of (i) CaHPO ₄ *2H ₂ 0 particles and (ii) particles of a calcium source comprising Ca (P0 ₄ ) ₂ 0 wherein said CaHP0 ₄ *2H ₂ 0 and said calcium source comprising Ca ₄ (P0 ₄ ) ₂ 0 are present in an amount sufficient to form phase-pure HAp having a Ca/P ratio of 1.5 to 1.67 within 1 hour. The dry particulate agglomerate composition can be used in preparing a bone-like substitute composition or for a dental composition for filling teeth. The dry particulate agglomerate composition forms phase pure

hydroxyapatite upon setting in a clinically relevant time in vivo while being biocompatible and possessing mechanical properties in the range of those exhibited by natural bone and tooth materials. The synthetic bone- like composition is also conducive to the rapid ingrowth of bone.

The present invention is also directed to an aqueous composition which comprises i. ) as a solids component (A) a dry particulate agglomerate composition which consists essentially of agglomerates, each of said agglomerates being a homogenous mixture of CaHP0 ₄ *2H _j O particles and particles of a calcium source comprising Ca ₄ (P0 ₄ ) ₂ 0, said agglomerate being prepared by mixing (a) CaHP0 ₄ *2H ₂ 0 particles with (b) particles of a calcium source comprising Ca (P0 ₄ ) ₂ 0 in (c) a non-aqueous liquid, and removing said non-aqueous liquid; said CaHP0*2H ₂ 0 and said calcium source comprising Ca ₄ (P0 ₄ ) ₂ 0 are present in an amount sufficient to form phase-pure hydroxyapatite having a Ca/P ratio of from 1.5 to 1.67; and

(B) a polymeric material which is capable of promoting mineralization of hydroxyapatite from said particles (A) and wherein the weight ratio of (A)/(B) is from 100/0 to 40/60, respectively: and (ii.)a physiologically acceptable aqueous liquid; said aqueous composition having a liquid-to-total solids weight ratio of 0.15 to 1.5: and wherein said aqueous composition reacts to form phase pure hydroxyapatite from component (A) at physiological temperatures within 1 hour, and a method of making said composition.

Another object of the present invention is to provide a physiologically useful hydroxyapatite composition comprising hydroxyapatite prepared from said dry particulate agglomerate composition as described above, and optionally a polymeric material capable of

promoting mineralization of hydroxyapatite, for repairing teeth or for bone grafting and implantation, wherein said coπposition meets the requirements of a bone-like material and which cure to form phase-pure hydroxyapatite in a surgically relevant time, preferably under 1 hour.

A further object of the present invention is to provide a method of filling in any bone defects with, or implanting a biocompatible and structurally strong, easily formed synthetic bone-like coπposition by using the dry particulate agglomerates of the present invention.

Still a further object of the present invention is to provide a method of making a particulate agglomerate composition capable of producing phase-pure hydroxyapatite without additional sources of calcium and/or phosphate in the presence of an aqueous liquid, and which, optionally, can be premixed with a polymeric material capable of promoting mineralization of hydroxyapatite prior to use in a body which comprises:

(a) mixing (i) CaHPO^H particles with (ii) particles of a calcium source comprising Ca ₄ (P0 ₄ ) ₂ 0, in a non-aqueous liquid wherein said CaHP0 ₄ *2H ₂ 0 and said calcium source comprising Ca ₄ (P0 ₄ ) ₂ 0 are present in an amount sufficient to form phase-pure hydroxyapatite having a Ca/P ratio of 1.5 to 1.67, to form said particulate agglomerates; and

(b) collecting said particulate agglomerates. Still a further object of the invention is to provide a kit comprising a container comprising a dry particulate agglomerate composition capable of producing phase-pure hydroxyapatite without additional sources of calcium and/or phosphate in the presence of an aqueous liquid consisting essentially of agglomerates, each individual agglomerate being a homogenous mixture of (i)

CaHP0 ₄ *2H ₂ 0 particles and (ii) particles of a calcium

source comprising Ca ₄ (P0) ₂ 0 wherein said CaHPCy2H ₂ 0 and said calcium source comprising Ca ₄ (P0 ₄ ) ₂ 0 are present in an amount sufficient to form phase-pure HAp having a Ca/P ratio of 1.5 to 1.67, which can be stored over long periods of time; and which can . be utilized for the preparation of a hydroxyapatite composition for a dental or surgical procedure which will benefit from using said hydroxyapatite composition.

This and other objects and advantages of this invention are described and are particularly delineated in the appended claims.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 is a phase diagram showing stability regions for calcium phosphates.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

One embodiment of the present invention is directed to a method of producing a particulate agglomerate composition, each of said particulate agglomerates being a homogenous mixture of (i) CaHP0 ₄ *2H ₂ 0 particles and (ii) particles of a calcium source comprising Ca ₄ (P0 ₄ ) ₂ 0, which are capable of forming hydroxyapatite in the absence of additional sources of calcium and/or phosphate, which comprises the steps of:

(a) mixing (i) CaHPCy2H ₂ 0 particles with (ii) particles of a calcium source comprising Ca ₄ (P0 ₄ ) ₂ 0 in a non-aqueous liquid, wherein said CaHP0 ₄ *2H ₂ 0 and said calcium source comprising Ca ₄ (P0 ₄ ) ₂ 0 are present in an amount sufficient to form phase pure hydroxyapatite having an overall Ca/P ratio of 1.5 to 1.67; and

(b) collecting said particulate agglomerates. By homogenous mixture is meant complete homogeneity of the CaHP0 ₄ *2H ₂ 0 particles and the particles of the

calcium source comprising Ca ₄ (P0 ₄ ) ₂ 0 in each individual particle capable of producing phase pure hydroxyapatite having a Ca/P ratio of 1.5 to 1.67. That is, the particles of CaHPO^IL and the particles of the calcium source comprising Ca (P0 ₄ ) ₂ 0 are . completely dispersed among each other within each individual agglomerate.

By particles of a calcium source comprising

Ca ₄ (P0 ₄ ) ₂ 0 is meant particles consisting of Ca ₄ (P0 ₄ ) ₂ 0 as the sole calcium source, or particles consisting essentially of Ca ₄ (P0 ₄ ) ₂ 0 and CaO, or particles consisting essentially of Ca ₄ (P0 ₄ ) ₂ 0 and Ca,(P0 ₄ ) ₂ .

The ol ratio of CaHP0 ₄ *2H ₂ 0 to Ca ₄ (P0 ₄ ) ₂ 0 sufficient to form phase-pure HAp having a Ca/P ratio of 1.5 to 1.67 is 2/1 to 1/1, respectively, when the sole calcium source is Ca ₄ (P0 ₄ ) ₂ 0.

When CaO or Ca ₃ (P0 ₄ ) ₂ is present with the Ca ₄ (P0 ₄ ) ₂ 0 as the calcium source, the upper limit in the mole ratio should be 0.2 (CaO or Ca ₃ (P0 ₄ ) ₂ / Ca ₄ (P0 ₄ ) ₂ 0).

For example, when the calcium source consists of CaO and Ca ₄ (P0 ₄ ) ₂ 0 in a mole ration of 0.2, then the mole proportion of CaHP0 ₄ *2H ₂ 0 to CaO to Ca ₄ (P0 ₄ ) ₂ 0 is 2.5:0.2:1, respectively, to produce HAp having a Ca/P of 1.5. When the calcium source consists of CaO and Ca ₄ (P0) ₂ 0 in a mole ration of 0.2, then the mole proportion of CaHP0 ₄ *2H ₂ 0 to CaO to Ca ₄ (P0 ₄ ) ₂ 0 is

1.38:0.2:1, respectively, to produce HAp having a Ca/P of 1.67.

For example, when the calcium source consists of Ca ₃ (P0 ₄ ) ₂ and Ca ₄ (P0 ₄ ) ₂ 0 in a mole ratio of 0.2, the mole proportion of CaHP0 ₄ *2H ₂ 0 to Ca ₃ (P0 ₄ ) ₂ to Ca ₄ (P0 ₄ ) ₂ 0 is 2:0.2:1, respectively, to produce HAp having a Ca/P of 1.5. When the calcium source consists of Ca ₃ (P0 ₄ ) ₂ and Ca ₄ (P0 ₄ ) ₂ 0 in a mole ratio of 0.2, the mole proportion of

CaHP0 ₄ *2H ₂ 0 to Ca ₃ (P0 ₄ ) ₂ to Ca ₄ (P0 ₄ ) ₂ 0 is 0.88:0.2:1, respectively, to produce a HAp having a Ca/P ratio of 1.67.

While not being bound to any theories, important advantages of preparing the inventive particulate agglomerate composition, wherein each particulate agglomerate is a homogenous mixture of CaHP0 ₄ *2H ₂ 0 particles and particles of a calcium source comprising Ca ₄ (P0 ₄ ) ₂ 0, which are capable of forming phase pure hydroxyapatite, of the present invention in a non- aqueous liquid are the following. The presence of a non-aqueous liquid enhances the mixing by affecting the surface charges on the particles allowing a much higher degree of mixing than can be achieved by dry blending, dry mixing or dry grinding of the mixture.

After the inventive particulate agglomerate composition is achieved, the non-aqueous liquid is removed from the particles by, e.g., filtration. The process of the final liquid removal is by evaporation.

By way of explanation of the prior art, all acidic calcium phosphates and Ca ₄ (P0 ₄ ) ₂ 0 dissolve incongruently. This means that when a crystal of CaHP0 ₄ or Ca(H ₂ P0 ₄ ) ₂ .H ₂ 0 dissolves, it creates a solution which is supersaturated in another calcium phosphate before the solution becomes saturated with respect to the dissolving crystal (see Figure 1) . This means that the other calcium phosphate precipitates on the surface of the dissolving crystal. For CaHP0 ₄ , CaHP0 ₄ .2H ₂ 0 and for Ca ₄ (P0 ₄ ) ₂ 0, the other calcium phosphate is hydroxyapatite. For Ca(H ₂ P0 ₄ ) ₂ .H ₂ 0, the other calcium phosphate is CaHP0 ₄ 2H ₂ 0. Therefore, when these calcium phosphates are used as reactants for hydroxyapatite, their dissolution causes hydroxyapatite to form on their surfaces, reduces the effective surface area for dissolution, and slows down reaction.

However, when CaHP0 ₄ *2H ₂ 0 particles and particles of a calcium source comprising Ca ₄ (P0 ₄ ) ₂ 0 are mixed with the intimacy achieved by using a non-aqueous liquid to form particulate agglomerate composition as in the present invention, this effect is minimized. Such an inventive

particulate agglomerate composition cannot be formed by dry intergrinding or dry mixing as in the prior art. The inventive particulate agglomerate composition must be formed by mixing in the presence of a non-aqueous liquid. Since the produced particulate agglomerates do not contain protons in the form of H _j P0 ₄ which could initiate further reactivity, the dry particulate agglomerate composition can be stored for an unlimited period of time, if kept dry. The extended shelf-life of such a product contributes to its advantage as a dental or bone-like substitute which can be constituted in an operating theater in a surgically relevant timeframe.

The time of mixing in the non-aqueous liquid is not critical so long as the time is sufficient to provide agglomerates, wherein each agglomerate is a homogeneous mixture of the CaHP0 ₄ *2H ₂ 0 particles and particles of a calcium source comprising Ca ₄ (P0 ₄ ) ₂ 0, and wherein the individual agglomerates can form phase-pure hydroxyapatite within 1 hour, and particularly within 30 minutes in the presence of an aqueous liquid.

The dry particulate agglomerate composition of the present invention can be further mixed with a polymeric material which acts as a substrate for hydroxyapatite mineralization and can be further sterilized by heat, by gamma radiation or by ethylene oxide.

Formation of hydroxyapatite in vivo from the particulate agglomerate composition of the present invention allows the reactants to be placed in a very close association with the bone or tooth being repaired. This cannot be achieved by using a preformed hydroxyapatite prosthesis.

Hydroxyapatite Formation - As an example of using the dry particulate agglomerate composition of the present invention, calcium deficient hydroxyapatite (having a Ca/P ratio of 1.5) can be formed from the intimate mixture of particles by the following reaction:

EQ [ 1 ]

H ₂ 0

6CaHP0 ₄ *2H ₂ 0+3Ca ₄ ( P0 ₄ ) ₂ 0 3Ca,HP0 ₄ ( P0 ₄ ) ₅ OH+13H ₂ 0

To prepare the inventive particulate agglomerate composition, the mixing must take place in a non- aqueous liquid. Preferably the non-aqueous liquid is of sufficient volatility so as to be easily removed by evaporation after the formation of the intimate mixture of particles. The non-aqueous liquid is not a solvent for said CaHP0 ₄ *2H ₂ 0 or Ca ₄ (P0 ₄ ) ₂ 0. Moreover, the non-aqueous liquid does not react directly with the CaHP0 ₄ *2H ₂ 0 particles and particles of a calcium source comprising Ca ₄ (P0 ₄ ) ₂ 0. When it is desired to produce only hydroxyapatite complete at reaction, the mole ratio amount of said CaHP0 ₄ *2H ₂ 0 and said calcium source comprising Ca ₄ (P0 ₄ ) ₂ 0 are selected to achieve Ca/P ratios from 1.5 to 1.67.

The amount of non-aqueous liquid used in preparing the inventive individual particles of the present invention is not critical. An amount of 1 volume of reactants to 2 volumes of non-aqueous liquid may be used as a guideline. Moreover, the non-aqueous liquid does not have to be non-toxic since the non- aqueous liquid is completely removed from the inventive individual particles prior to use in vivo .

By non-aqueous liquid is meant, any liquid which will not dissolve the CaHP0 ₄ *2H ₂ 0 particles or the particles of the calcium source comprising Ca ₄ (P0 ₄ ) ₂ 0, will not react with either type of the particles, and will not allow the particles to react with each other.

Examples of such non-aqueous liquids include, but are not limited to, liquid aliphatic hydrocarbons which may be straight, branched or cyclic, and aromatic hydrocarbons which may be further substituted with functional groups such as halogens. Examples of

liquid aliphatic hydrocarbons, include, but are not limited to substituted or unsubstituted liquid alkanes, and liquid alkenes. Examples include but are not limited to butane, pentane, hexane, heptane, CC1 ₄ , CH ₂ C1 ₂ and C ₂ Cl _β .

The temperature range for reacting Ca ₄ (P0 ₄ ) ₂ 0 with

CaHP0 ₄ *2H ₂ 0 is not critical, so long as the temperature is below the decomposition temperature of the CaHP0 ₄ *2H ₂ 0. The particle size of the CaHP0 ₄ *2H ₂ 0 particles and Ca ₄ (P0) ₂ 0 particles are in the range of from 0.05 to 10 μm, and preferably, 0.05 to 5 μm.

The average particulate agglomerate size is in the range of 1 μm to 15 μm. Figure 1 shows the compositional relationships between CaHP0 ₄ and HAp to be peritectic reactions. As a result, the hydrolysis of CaHP0 ₄ results in it being overgrown by hydroxyapatite or intermediate products and causing the complete reaction to hydroxyapatite to become undesirably diffusionally controlled. For example, the hydrolysis of CaHP0 ₄ occurs as follows:

H ₂ 0

9CaHP0 ₄ → Ca ₉ (HP0 ₄ ) ( P0 ₄ ) ₅ OH + 6H ^* + 3HP0 ₄ 2- EQ [2 ]

even in the presence of Ca ₄ (P0 ₄ ) ₂ 0. The hydroxyapatite formed does so as a coating on the CaHP0 ₄ reactant and results in a lengthy period of time to complete reaction. A similar set of events occur when one of the initial reactants is CaHP0 ₄ *2H ₂ 0 except when incorporated into the particulate agglomerate composition of the present invention. CaHP0 ₄ cannot be used in the inventive particulate agglomerate coπposition as shown in the Comparative Experiment which follows.

According to the present invention mixing of CaHP0 ₄ *2H ₂ 0 with a calcium source comprising Ca ₄ (P0 ₄ ) ₂ 0 in a non-aqueous liquid results in the formation of particulate agglomerates, each agglomerate of which is a homogenous mixture of Ca ₄ (P0 ₄ ) ₂ 0 and a calcium source comprising CaHP0 ₄ *2H ₂ 0. When the particulate agglomerate composition of the present invention is mixed with a physiologically acceptable aqueous liquid such as sterile water for injection, or normal saline for injection, these combinations of reactants react completely to form hydroxyapatite within minutes to an hour without the formation of intermediates. Preparing the inventive particulate agglomerate composition with a non-aqueous liquid is required to obtain the degree of intimacy needed to avoid the extended periods of reaction caused by such overgrowth of HAp.

The type of reaction vessel or its size for preparing the particulate agglomerate coπposition of the present invention is not critical, and can be any vessel capable of holding the CaHP0 ₄ *2H ₂ 0, Ca ₄ (P0 ₄ ) ₂ 0 and non- aqueous liquid so as to allow the mixing to take place. An example of such a vessel is any commercially available polyethylene bottle. Examples of pellets to facilitate mixing of the individual particles include, but are not limited to, zirconia, alumina and polycarbonate pellets.

The present invention was developed because mechanical mixtures of dry calcium sources and acidic phosphate source e.g., CaHP0 ₄ and Ca ₄ (P0 ₄ ) ₂ 0, or Ca ₄ (P0 ₄ ) ₂ 0 and a calcium source comprising CaHP0 ₄ *2H ₂ 0 in suitable proportions, to form hydroxyapatite as shown in U.S. Patent 4,612,053 to W. Brown and Chow requires an extended period of time to reach completion while those of Ca(H ₂ P0 ₄ ) ₂ H ₂ 0, H ₃ P0 ₄ or H ₃ P0 ₄ .1/2H ₂ 0 with Ca ₄ (P0 ₄ ) ₂ 0 form intermediates which may also persist for lengthy

periods, which is undesirable in the average time for a dental or surgical procedure.

When hydroxyapatite is produced according to the present invention and is used to repair dentitia, the inventive particulate agglomerate composition can be used alone, or additional components can be mixed therewith to alter the physical properties of the produced hydroxyapatite.

When the particulate agglomerate composition of the present invention is used to produce a synthetic bone¬ like HAp composition, polymeric materials capable of allowing mineralization of hydroxyapatite can be mixed therewith in an amount sufficient to achieve any of a number of desired effects. Such desired effects can be, e.g., bone formation, enhanced resorption, growth of osteoblasts and/or osteoclasts, angiogenesis, cell entry and proliferation, mineralization, altering mechanical properties, or the like.

In defining the aqueous composition embodiment of the invention, by solids component is meant the total weight of (A) inventive particulate agglomerate composition; or the total weight of (A) plus the polymeric material capable of promoting mineralization of hydroxyapatite (B) . The weight ratio of (A) to (B) is in the range of 100/0 to 40/60 respectively, particularly 99.99/0.01 to 60/40 respectively, and more particularly 99/1 to 60/40 respectively, depending on the polymeric material employed. When the polymeric material is bone associated protein, and particularly treated insoluble collagen, the weight ratio of (A)/(B) is 99/1 to 60/40.

By liquid-to-total solids weight ratio is meant the ratio of the total weight of the physiologically acceptable aqueous liquid to the total weight of the solids present in the composition. The total weight of the solids will include the solids component described

in the preceding paragraph as well as any additional additives, such as fillers, bisphosponates, etc.

The physiologically acceptable aqueous liquid which is used to constitute formation of phase pure hydroxyapatite in vivo will be substantially pure, e.g., sterile water for injection, normal saline for injection or equivalents thereof.

Examples of such polymeric materials (B) can be synthetic polymers, or proteins known to be associated with bone, cartilage, or dentin.

With respect to synthetic polymeric materials those which contain functional groups capable of being hydrolyzed by H ₂ 0, such as carboxyl, amine or sulfonate groups, are useful so long as the functional groups on the polymer are capable of forming salt bridges with calcium and phosphate. Examples of water soluble synthetic polymers, include but are not limited to polyacrylic acid and polyacrylic-itaconic acid co- polymers. Examples of such polymeric bone proteins include collagen, particularly e 1, osteonectin ZHS- glycoproteins, sialoproteins (BSP) , bone-Gla-protein (BGP) matrix-Gla-protein bone proteoglycan bone phosphoglycoprotein, bone phospho-protein, skeletal growth factor, and the like.

Examples of such polymeric cartilage proteins include chondrocalcining proteins.

Examples of such polymeric dentin or enamel proteins include glycoproteins such as Gla protein, phosphophoryn, amelogelnin, and enamelin.

A particularly preferred polymeric material is insoluble collagen which has been treated and which is prepared as follows.

Collaσen Treatment Process - Collagen treatment serves three functions in contributing to the development of hydroxyapatite-collagen bone replacement composites. It allows coarse collagen granules to be

converted into fibrils. It orients these fibrils in a three dimensionally interconnected network. This network structure provides a template on which hydroxyapatite formation occurs. Thus, the treated collagen prepared by the process of the present invention also serves to facilitate hydroxyapatite formation in vivo.

The collagen treatment method is achieved by a combination of physical, chemical and mechanical actions. Granular insoluble collagen obtained from, e.g., Sigma Chemical Company, is suspended in an acidic solution and the collagen subjected to a mechanical action, such as from a commercial blender (step a) . The organic acid breaks the hydrogen bonds between fibrils and allows their expansion in solution by hydration. The acid may also be selected to control the rate of hydroxyapatite formation (discussed below) . Mechanical shear promotes the physical separation and expansion of the fibrils. After this processing step, the suspension is rapidly frozen in liquid nitrogen (step b) . Freezing in other low boiling point liquids (He, H ₂ , 0 ₂ , Ar, etc., or solids (C0 ₂ ) will also work. Expansion of water present on freezing this suspension assists in the expansion and separation of the dense tendinous collagen structure. The frozen mass is then thawed and mechanically sheared for a second time to further the separation of the fibrils (step c) . This process can be repeated as many times as is necessary.

After reblending, the suspension is again frozen using liquid nitrogen and then freeze-dried. Freeze drying allows the removal of both the water and the organic acid. This process results in a three dimensional organic matrix consisting of oriented submicron fibrils interconnected with fibrils and collagenous membranes. This matrix is a result of the freezing direction, namely the fibrils align in the direction of the solidification front. The extended

structure provides a network for the formation of hydroxyapatite. This structure has pores and channels ideal for the incorporation of the hydroxyapatite. In addition, the pore and channel dimensions can be easily controlled by altering the acid concentration and water content in step (a) . Thus, altering the acid concentration and water content to control the collagen microstructure can also control the resulting properties of the synthetic bone. The treated collagen has pore diameters in the range of from 2 μ to 100 μm.

A number of features are associated with treating insoluble collagen. First, the insoluble collagen can be processed quite rapidly. Insoluble collagen harvested by known surgical methods from another part of the individual to be treated can be used as a result. Collagen obtained from another individual can also be treated.

Other materials may be added to the aqueous composition of the present invention to provide for specific types of mechanical physical properties. Both inorganic and organic fibrous materials can be employed. Examples include hydroxyapatite fibers and biodegradable organic fibers such as polyglycolic acid.

Fillers can be mixed with the inventive particulate agglomerate composition and, optionally, polymeric materials prior to reaction with physiologically acceptable aqueous liquid, in order to control the porosity, and increase the early strength of the compositions during mixing and surgery. Examples of fillers include preexisting HAp, CaHP0 ₄ , CaHP0 ₄ coated with fluorapatite, Ca ₄ (P0 ₄ ) ₂ 0 coated with CaHP0 ₄ . The filler can be formulated to any particle size, preferably between 15 to 75 μm. The proportion of filler, when present, can be as high as 65 wt% based on the weight of the solids component (A) and (B) of the composition.

Further, additional additives can be added which include, but are not limited to fluoride sources, antibiotics, surfactants, and dispersants in order to exert their intended effect. With respect to the addition of antibiotic, the choice of antibiotic incorporated into the coπposition depends on the microorganisms to be treated, and is well within the level of skill in the art.

Effective amounts of antibiotics can be incorporated into the compositions in order to prevent or treat infections of the bone or dentitia or infections of surrounding tissue. Effective amounts are well known or can be readily ascertained by one skilled in the art. Examples of antibiotics include, but are not limited to penicillin, cephalothin, tobramycin, gentamycin, nafcillin, rifampin, clindamycin, polymyxin, metronidazole, chloramphenicol well known in the art as evidenced by the Physicians Desk Reference, 48th Edition, 1994.

Effective amounts of the antibiotics can be dissolved in the physiologically acceptable liquid or can be mixed with the inventive particulate agglomerate coπposition and, optionally, the polymeric material. The rate of release of antibiotics can be controlled by controlling the initial concentration of the antibiotic.

A hydroxyapatite tooth or bone substitute composition prepared by the present invention can be prepared by a number of methods. In a first method, the polymeric material capable of promoting mineralization of hydroxyapatite can be added directly into the non-aqueous liquid used in forming the inventive particulate agglomerate composition prior to removal of the non-aqueous liquid. In a second method, the inventive particulate agglomerate composition may be mixed with a polymeric material capable of promoting mineralization of

hydroxyapatite in an aqueous phase, prior to use in vivo.

When preparing the coπposition of the present invention, the liquid-to-total solids weight ratio is from 0.15 to 1.5 (liquid-to-total solids component), preferably 0.25 to 1.0.

The additives can be added at any time during the process of preparing the aqueous composition, up until use of the coπposition in vivo in the relevant procedure.

Regardless of which method is used, the resultant composition can be utilized in mass as a monolith or can be manipulated with a polymeric material, such as collagen, to achieve a net anisotropic orientation. In preparing the particulate agglomerate composition of the present invention for use in vivo, if the first method is used, the non-aqueous liquid is removed by evaporating at ambient temperature. If the second method is used, a blend of the inventive particulate agglomerate coπposition and polymeric material in aqueous phase is frozen in liquid nitrogen and then freeze dried. After the non-aqueous liquid or the frozen water is removed, the resulting blend is crushed lightly to yield a slightly fibrous powder. This powder can then be sterilized and stored dry ready for constitution during the relevant procedure. When needed, it can be hand or mechanically mixed with 150% of its weight of a physiologically acceptable liquid, such as sterile water for injection, or normal saline. This mixing step occurs prior to anticipation of use in vivo.

Compositional Ad-i s ments - Sodium and carbonate can be incorporated into the compositions by mixing CaHP0 ₄ *2H ₂ 0 and a calcium source comprising Ca ₄ (P0 ₄ ) ₂ 0 described above in the presence of up to 7% by weight of particulate NaHC0 ₃ , based on the weight of the particles

of an intimate mixture of CaHP0 ₄ *2H ₂ 0 and a calcium source comprising Ca ₄ (P0 ₄ ) ₂ 0 to obtain a composition which is as close as possible to natural bone and/or teeth. The incorporation of carbonate and sodium into the hydroxyapatite composition demonstrates that the Na ^* and C0 ² ₃ - concentrations in solution decreased linearly as the formation of hydroxyapatite proceeds, attaining concentrations of a few millimoles at complete reaction. Use of 7% by weight of NaHC0 ₃ based on the weight of the particles of HAp resulted in a hydroxyapatite coπposition containing approximately 3.6 wt% C0 ₃ . As a result of incorporating up to 7% by weight of Na and carbonate and particularly, 1 to 7% by weight of Na and carbonate, a hydroxyapatite composition close to that of native bone can be formed by a reaction which is physiologically compatible in vivo. Therefore, a mineral phase close in composition to that of natural bone and teeth can be formed in vivo. Developing prostheses which will remodel in vivo in a manner similar to that of natural bone and teeth.

A variety of hydroxyapatite compositions may be achieved, depending on desired application of the dentist or surgeon. Alkali phosphates can be added to the aqueous solution to influence the kinetics of reactant dissolution, for example by making such dissolution increasingly congruent. A fluoride source, for example, NaF or CaF ₂ , can be added to form fluoride substituted apatite in which can be present to partially or completely substitute for OH. Moreover, various acids, e.g., acetic acid and citric acid can be added to the aqueous phase, which do not enter the apatite structure, but which influence the rate of hydroxyapatite formation. Specifically, small additions, e.g., (from 15 to 150mM) of acetic acid to the aqueous phase accelerate the onset of hydroxyapatite formation while small additions of citric acid (from 15 to 150 M) delay the onset of hydroxyapatite formation.

Such additives provide the means to control the rate of hydroxyapatite formation in vivo. The presence of such additives may be synergistically associated with the presence of the processed collagen (as discussed) . Having generally described the invention, the following specific examples are included for the purpose of illustration only and not intended to limit the scope of the invention. For ease of calculation, the liquid components in the following examples are rounded off to have a density of 1.0 g/ml. All liquid-to-total solids ratios are by weight.

EXAMPLE 1 Preparation of a Particulate Agglomerate Composition

Capable of Forming HAp having a Ca/P Ratio of 1.5

(calcium deficient HAp)

In a typical run to prepare a particulate agglomerate coπposition which will be employed to form Ca,HP0 ₄ (P0 ₄ ) ₅ OH (HAp having a Ca/P ratio of 1.5) in vivo,

172 g of CaHP0 ₄ *2H _j O (Fisher Chemical Co.) having a particle size of from 0.5 μm to 5 μ is mixed with 183 g of Ca ₄ (P0 ₄ ) ₂ 0 having a particle size of from 0.5 μm to 5 μm. The Ca ₄ (P0 ₄ ) ₂ 0 is prepared by firing an intimate mixture of equimolar amounts of CaC0 ₃ and CaHP0 ₄ powders to 1400°C for 4 hours. After cooling the Ca ₄ (P0 ₄ ) ₂ 0 is ground to a particle size of 0.5 μ to 5 μm using standard powder preparation techniques. This material is then mixed with CaHP0 ₄ *2H ₂ 0 which has been ground to having a particle size of from 1 μm to 5 μm using standard powder preparation techniques, by placing it and Ca ₄ (P0 ₄ ) ₂ 0 in a 1-liter polyethylene bottle with approximately 400 ml of heptane.

Approximately 250g of zirconia or alumina, or lOOg polycarbonate pellets are added and the bottle is

sealed. The presence of the pellets facilitate agglomerate formation. The bottle is then placed in any shaking or rolling device.

There is a negligible reduction in particle size during reaction in heptane; therefore, this intimate mixing step does not involve grinding. The particulate agglomerate coπposition is formed according to the present process and cannot be duplicated by grinding in air, grinding in aqueous solvents, or grinding in water. After mixing, the slurry of particulate agglomerates and the pellets is placed on a filter funnel and the bulk of the heptane is removed by vacuum suction and can be reused after distillation. The pellets are then removed. Any material which adheres to pellets is removed by washing in heptane. Heptane not removed by vacuum suction is allowed to evaporate, either at room or slightly elevated temperature. Evaporation may also be carried out in vacuum.

When reacted with a physiologically acceptable aqueous liquid at physiological temperature, the particulate agglomerate composition of the invention achieves a substantial degree of completion to HAp in 15 minutes or less and converts completely to HAp in periods of time which range from less than 30 minutes to less than 1 hour.

EXAMPLE 2 Preparation of Mixed Calcium Sources

Calcium sources comprising of a mixture of Ca ₄ (P0 ₄ ) ₂ 0 and CaO or of Ca ₄ (P0 ₄ ) ₂ 0 and Ca ₃ (P0 ₄ ) ₂ are prepared at high temperature by processing similar to that described in Example 1, above. This is accomplished by varying the proportions of calcium- containing and phosphate-containing reactants. Mixtures composed of Ca ₄ (P0 ₄ ) ₂ 0 and CaO require Ca/P ratios greater than 2 while mixtures of Ca ₄ (P0 ₄ ) ₂ 0 and Ca ₃ (P0 ₄ ) ₂ require ratios less than 2. Mixtures of Ca ₄ (P0 ₄ ) ₂ 0 and Ca ₁₀ (PO ₄ ) ₆ O

(oxyapatite) are prepared when the reaction temperature is maintained below about 1275°C. When the high temperature reaction time is shorter than about 4 hours and when the reaction temperature is below about 1300°C, mixtures of Ca ₄ (P0 ₄ ) ₂ 0, CaO, and Ca ₁₀ (PO ₄ ) _β O are prepared. Non-equilibrium mixtures of CaO and Ca ₃ (P0 ₄ ) ₂ are produced if the reaction temperature is above about 1600°C, which is high enough to attain a liquid phase.

The inventive particulate agglomerate composition can also prepared from these calcium sources by mixing them with CaHP0 ₄ *2H _j O particles in a similar fashion as set forth in Example 1. Of course, the proportions of the reactants must be adjusted to ensure that the particles formed have a Ca/P ratio in the range of 1.5 to 1.67 which can be readily ascertained by one skilled in the art.

EXAMPLE 3 Collagen processing Insoluble collagen granules (Sigma) are used as the starting material. Approximately 10 g of insoluble collagen granules are placed in 100 ml of solution of acetic acid (50 mM) . This mixture is allowed to sit for approximately 16 hours. The mixture is then mechanically sheared in a Waring commercial blender for 5 minutes. The mixture is then rapidly frozen in liquid nitrogen. Freezing assists the separation of the collagen fibers. The mixture is then allowed to thaw and refrozen in liquid nitrogen a second time. This process may be repeated as many times as is desired, but in the detailed example (to be described) it was done twice. After the last freezing cycle, the frozen mass is freeze dried. Freeze drying removes both water and acetic acid such that, if the collagen is immersed in water at a liquid-to-solid weight ratio of 1 after drying is complete, the pH does not drop below 5.5.

EXAMPLE 4 Composite formation

Collagen, treated as described above in Example 3 is mixed with the particulate agglomerate composition as described above in Example 1. Three proportions, expressed in terms of the hydroxyapatite:collagen volume ratios, of 2:1, 5:1 and 10:1 is prepared. A typical mix weighs 50 grams. The proportions of CaHP0 ₄ *2H ₂ 0 and Ca ₄ (P0 ₄ ) ₂ 0 used is selected to produce calcium deficient hydroxyapatite, Ca,HP0 ₄ (P0 ₄ ) ₅ OH, at complete reaction, see EQ [1] and Example I.

EXAMPLE 5 Production of HAp/Collagen Composition

Containing Tobramycin Sulfate

100 gm of the particulate agglomerate composition prepared according to Exaπple 1 is mixed with 4.7 gm of treated collagen prepared according to Example 3 (solid component) . A stock saline solution of tobramycin sulfate is prepared by dissolving 40 g tobramycin sulfate per ml of normal saline. 1.2 gm of the tobramycin sulfate-containing saline (liquid component) is added to 3.0 gm of the solid component to form a mixture (a liquid-to-total solids weight ratio of 0.4). The mixture is mixed for 2 minutes at room temperature and surgically implanted into a 15 mm cranial defect in a rabbit. The tobramycin sulfate-to HAp weight ratio is approximately 16 mg of tobramycin sulfate per gram of phase pure HAp.

EXAMPLE 6 Production of HAp Containing Tobramycin Sulfate

The particulate agglomerate coπposition is prepared according to Exaπple 1 in order to produce calcium deficient HAp (Ca/P = 1.5) (solids component). No treated collagen is used in this example. 1.02 gm of

the tobramycin sulfate stock saline solution prepared according to Example 7A is mixed with 3.0 gms of the solid component (a liquid-to-total solids weight ratio of 0.34) for two minutes at room temperature. The coπposition is surgically implanted in 15 mm cranial defects in a rabbit.

A wide range of rates of antibiotic release (designed to maintain a release rate in the therapeutic range of a selected period of time) can be achieved.

EXAMPLE 7 Production of HAp/Collagen + HAp-Coated CaHP0 ₄

Filler to Control Porosity Coating of CaHP0 ₄ filler particles with HAp is carried out as follows: 100 gm of CaHP0 ₄ particles having a particle size of 35 μ are placed in 400 ml of distilled water and stirred slowly. After about 16 hours, when the pH of the water reaches approximately 4.6, the produced HAp-coated CaHP0 ₄ filler particles are filtered from solution using filter paper and a Buchner funnel. These HAp-coated CaHP0 ₄ filler particles are then washed with approximately 100 ml of distilled water and dried in a desiccator over Drierite (CaS0 ₄ 'l/2H ₂ 0) .

At the time of surgery, 0.9 gm of normal saline solution is mixed with 1.2 gm of HAp-coated CaHP0 ₄ filler particles and with 1.8 gm of a mixture of solid components containing the particulate agglomerate coπposition prepared according to Exaπple 1 and treated collagen prepared according to Example 3 wherein said mixture is obtained by mixing 100 gm of the particulate agglomerate composition of Example 1 and 4.7 gm of treated collagen of Example 3. The liquid-to-total solids (solids component + filler) weight ratio is 0.30. Mixing of the liquid and the total solids is carried out for 2 minutes at room temperature. The resultant composition is surgically implanted in a 15 mm cranial defect in a rabbit.

EXAMPLE 8

Production of HA /Collagen + FAp-Coated CaHP0 ₄

Filler to Control Porosity and Delivery Fluoride

Coating of CaHP0 ₄ filler particles with fluoroapatite (FAp) is carried out as follows: 200 gm of CaHP0 ₄ particles having a particle size of 35 μm are placed in 1000 ml of distilled water to which 0.44 gm of NaF has been added and stirred slowly for 16 hours at room temperature. The produced FAp-coated CaHP0 ₄ filler particles are filtered from solution using filter paper and a Buchner funnel. These FAp-coated CaHP0 ₄ filler particles are then washed with approximately 200 ml of distilled water and dried in a desiccator over Drierite (CaS0 l/2H ₂ 0) . At the time of surgery, 0.9 gm of normal saline solution is mixed with 1.2 gm of FAp-coated CaHP0 ₄ filler particles and with 1.8 gm of a mixture of solid components containing the particulate agglomerate composition according to Example 1 and treated collagen prepared according to Example 3 wherein said mixture is obtained by mixing 100 gm of the particulate agglomerate composition of Exaπple 1 and 4.7 gm of treated collagen of Example 3. The liquid-to-total solids (solids component + filler) weight ratio is 0.30. Mixing of the liquid and the total solids is carried out for 2 minutes at room temperature. The resultant coπposition is surgically implanted in a 15 mm cranial defect in a rabbit.

EXAMPLE 9

HAp/Collagen + HAp-Coated Ca ₄ (P0 ₄ ),0 Filler to Control Porosity

Coating of 57 μm Ca ₄ (P0 ₄ ) ₂ 0 particles (TetCP) with

CaHP0 ₄ is carried out as follows: 57.6 gm of 85% H ₃ P0 ₄ solution are mixed with approximately 400 ml of distilled water and heated to 80°C. 412 gm of 57 μm

Ca ₄ (P0 ₄ ) ₂ 0 particles are rapidly added to the H ₃ P0 ₄ solution while stirring. The solution is stirred for 4 hours at 80°C after which the particles are filtered from this 80°C solution using filter paper and a Buchner funnel. These CaHP0 ₄ -coated Ca ₄ (P0 ₄ ) ₂ 0 filler particles are then dried in a desiccator over Drierite (CaS0 ₄ ^' l/2H,0) .

At the time of surgery, 0.9 gm of normal saline solution is mixed with 1.2 gm of CaHP0 ₄ -coated TetCP filler particles and with 1.8 gm of a mixture of solid components containing the particulate agglomerate coπposition prepared according to Example 1 and treated collagen prepared according to Example 3 wherein said mixture is obtained by mixing 100 gm of the particulate agglomerate composition of Example 1 and 4.7 gm of treated collagen of Example 3. The liquid-to-total solids (solids component + filler) weight ratio is 0.30. The liquid used is normal saline. Mixing of the liquid and the total solids is carried out for 2 minutes at room temperature. The resultant composition is surgically implanted in a 15 mm cranial defect in a rabbit.

In contrast to the CaHP0 ₄ filler (Ca/P = 1) of Example 8 which is acidic, the composite TetCP/CaHP0 ₄ filler Ca/P = 1.8) used in this experiment is basic. The Ca/P ratio of 1.8 is selected because this ratio approximates the ratio in bone. This ratio is also selected because HAp formation at this ratio will theoretically take up carbonate. This composite filler has the theoretical capacity to form HAp by reaction in physiological fluids without added water. As a consequence, the implant should contain a lower proportion of porosity when the filler present has reacted to form HAp.

EXAMPLE IP

HAp/Collagen + HAp-Coated Ca ₄ (P0 ₄ ),0

Filler to Control Porosity The above experiment in Example 9 is repeated except the proportions of constituents are changed to: 0.9 gm of normal saline solution is mixed with 1.8 gm of CaHP0 ₄ -coated TetCP filler particles and with 1.2 gm of a mixture of solid components containing the particulate agglomerate coπposition prepared according to Example 1 and treated collagen prepared according to Example 3 wherein said mixture is obtained by mixing 100 gm of the particulate of Example 1 and 4.7 gm of treated collagen of Example 3. The liquid-to-total solids (solids component + filler) weight ratio is 0.30. The liquid used is normal saline. Mixing of the liquid and the total solids was carried out for 2 minutes at room temperature. The resultant composition is surgically implanted in a 15 mm cranial defect in a rabbit.

EXAMPLE 11 Preparation of a Particulate Agglomerate Composition

Capable of Forming HAp having a Ca/P Ratio of 1.67 (stoichiometric HAp) In a typical run to prepare a particulate agglomerate coπposition which will be employed to form Ca _I0 (PO ₄ ) _β (OH) ₂ (stoichiometric HAp) in vivo, 172 g of CaHP0 ₄ *2H ₂ 0 is mixed with 366 g of Ca ₄ (P0 ₄ ) ₂ 0. The Ca ₄ (P0 ₄ ) ₂ 0 is prepared by firing an intimate mixture of equimolar amounts of CaC0 ₃ and CaHP0 ₄ powders to 1400°C for 4 hours. After cooling the Ca ₄ (P0 ₄ ) ₂ 0 is ground to an average particle size of 0.5 μm to 5 μm using standard powder preparation techniques. This material is then mixed with CaHP0 ₄ *2H ₂ 0 which has been ground to having an average particle siz≤ of from 1 μm to 5 μm using standard powder preparation techniques, by placing it

and Ca ₄ (P0 ₄ ) ₂ 0 in a 1-liter polyethylene bottle with approximately 400 ml of heptane.

Approximately 325g of zirconia or alumina, or 13Og polycarbonate pellets are added and the bottle is sealed. The presence of the pellets facilitates mixing. This bottle is then placed in any shaking or rolling device. The produced intimate mixture of CaHP0 ₄ *2H _j 0 and a calcium source comprising Ca ₄ (P0 ₄ ) ₂ 0 particles can then react rapidly in the presence of water to form hydroxyapatite in vivo.

After mixing, the slurry of particulate agglomerates and the pellets is placed on a filter funnel and the bulk of the heptane is removed by vacuum suction which can be reused after distillation. The pellets are then removed by hand picking. Any material which adheres to pellets is removed by washing in heptane. Heptane not removed by vacuum suction is allowed to evaporate, either at room or slightly elevated temperature. Evaporation may also be carried out in vacuum.

3 grams of the obtained particulate agglomerate composition is mixed with 1.2 gm of normal saline solution for 2 minutes at room temperature. The liquid- to-total solids weight ratio is 0.4. The resultant composition is surgically implanted in a 15 mm cranial defect in a rabbit.

EXAMPLE 11A

The above experiment in Example 11 is repeated except that the liquid-to-total solids weight ratio was reduced to 0.36. The mixture is implanted in a 15 mm cranial defect in a rabbit.

EXAMPLE 12 In this experiment, a 1.8 gm of the particulate agglomerate coπposition prepared according to Example 11 is mixed with 1.2 gm of FAp-coated CaHP0 ₄ filler prepared

according to Exaπple 8, and 0.75 gm normal saline (a liquid-to-total solids weight ratio of 0.25). The mixture is implanted in a 15 mm cranial defect in a rabbit.

EXAMPLE 13 HAp + HEDP Bisphosphonates are of interest as therapeutic agents to control the rate of bone resorption and the rate of tooth decay. A bisphosphonate, HEDP (1- hydroxyethylidene-1, 1-diphosphonic acid) C ₂ H,P ₂ 0 ₇ , saline stock solution was prepared by mixing 2.5 gm of HEDP with 1000 ml of normal saline solution. 1.2 gm of the HEDP-containing saline solution is mixed with 3 grams of the particulate agglomerate composition prepared according to Exaπple 1. The liquid-to-total solids weight ratio is 0.4. The resultant coπposition is surgically iπplanted in 15 mm cranial defects in a rabbit. The HEDP-to-HAp weight ratio can be adjusted in three ways: (1) a larger amount of HEDP can be added to the saline solution, (2) the liquid-to-solids ratio can be increased, (3) the HEDP powder can be intermixed with the intimate mixture of particles prior to the addition of the liquid.

COMPARATIVE EXPERIMENT The rates of reaction for mixing CaHP0 ₄ *2H ₂ 0 or CaHP0 ₄ were determined when mixed to form calcium deficient HAp and stoichiometric HAp according to the following reactions:

Exs. 1A & IB. 2CaHP0 ₄ 2H ₂ 0 + 2Ca ₄ (P0 ₄ ) ₂ 0 => Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ + 4H.0

(calcium deficient HAp, Ca/P = 1.5) Exs. 2A & 2B. 6CaHP0 ₄ + 3Ca ₄ (P0) ₂ 0 =>

2Ca, ( HP0 ₄ ) ( P0 ₄ ) ₅ 0H + H ₂ 0

(calcium deficient HAp, Ca/P = 1.5)

Exs. 3A & 3B. 6CaHP0 ₄ 2H ₂ 0 + 3Ca ₄ (P0 ₄ ) ₂ 0 -> 2Ca,(HP0 ₄ ) (P0 ₄ ) ₅ OH + 12H ₂ 0

(stoichiometric HAp, Ca/P = 1.67) Exs. 4A & 4B. 2CaHP0 ₄ + 2Ca ₄ (P0 ₄ ) ₂ 0 => Ca ₁₀ (PO ₄ ) ₆ (OH) ₂

(stoichiometric HAp, Ca/P = 1.67)

The above reactants were mixed in two ways: (1)

(Exs. 1A, 2A, 3A and 4A) dry intergrinding of the powdered CaHP0 ₄ *2H ₂ 0 and Ca ₄ (P0 ₄ ) ₂ 0 or CaHP0 ₄ ; and (2) (Exs. IB, 2B, 3B and 4B) mixing CaHPO^H _j O with Ca ₄ (P0 ₄ ) ₂ 0 or

CaHP0 in the non-aqueous liquid heptane, followed by removing the non-aqueous liquid by filtration and its evaporation. Thus, a total of 8 experiments were carried out to determine the rate of HAp formation when these combinations of reactants were mixed with water at

37.4°C: formation of stoichiometric and calcium deficient HAp using mixtures of CaHP0 ₄ *2H ₂ 0 or CaHP0 ₄ with Ca ₄ (P0 ₄ ) ₂ 0 when the mixtures were obtained by dry intergrinding or by mixing in the presence of a non- aqueous liquid. The only instance where rapid reaction to HAp occurred was when CaHP0 ₄ *2H ₂ 0 and a calcium source comprising Ca ₄ (P0 ₄ ) ₂ 0 were mixed in a non-aqueous fluid. Rapid reaction occurred regardless of whether they were mixed in the proportion needed to produce calcium deficient HAp (reaction 1 above) or to make stoichiometric HAp (reaction 3 above) . Rapid reaction resulted in the formation of hydroxyapatite in 1 hour or less at 38°C. In the other cases, complete reaction was not achieved within 24 hours. The results appear in the Table below.

TABLE FORMATION OF HAp