Carbonated hydroxyapatite compositions and uses

Carbonated hydroxyapatite compositions and their preparation are described. The compositions are biologically resorbable and are prepared as flowable masses which can be administered by syringe to set in situ to serve as a support structure, filler, prosthesis or the like. Optionally the compositions may include proteins or serve as a depot for compositions of phrarmacological interest.

INTRODUCTION 
1. Technical Field 
The field concerns the preparation of substantially pure compositions of 
carbonate-substituted forms of hydroxyapatite, and the biomedical use of 
such compositions. 
2. Background 
A number of calcium phosphate minerals, such as hydroxyapatite, 
fluorapatite, octacalcium phosphate, whitlockite, brushite and monetite 
may have application as biocompatible materials. The various crystalline 
forms of the calcium phosphate minerals can confer different physical 
properties that may be more or less desirable for a specific biomedical 
application. For instance, octacalcium phosphate and whitlockite are less 
resorbable than brushite or monetite (Brown and Chow, Ann. Rev. of 
Materials Science (1976) 6:213-236). 
Of particular interest are the apatites. Apatite is a general term for a 
wide range of compounds represented by the general formula M.sup.2+.sub.10 
(ZO.sub.4.sup.3-).sub.6 Y.sup.-.sub.2, where M is a metal atom, 
particularly an alkali or alkaline earth atom, ZO.sub.4 is an acid 
radical, where Z may be phosphorous, arsenic, vanadium, sulphur, silicon, 
or may be substituted in whole or in part by carbonate (CO.sub.3.sup.2-), 
and Y is an anion, usually halide, hydroxy, or carbonate. When 
ZO.sub.4.sup.3- is partially or wholly replaced by trivalent anions (such 
as CO.sub.3.sup.2-) and/or Y.sup.- is partially or wholly replaced by 
divalent anions, then charge balance may be maintained in the overall 
structure by additional monovalent cations (such as Na.sup.+) and/or 
protonated acid radicals (such as HPO.sub.4.sup.2-). 
Hydroxyapatite (HAp), as well as its various forms, has been the focus of 
substantial interest because it is a major structural component of 
biological tissues such as bone, teeth, and some invertebrate skeletons. 
There are many situations where bone has been broken, surgically removed, 
destroyed, degraded, become too brittle, or been subject to other 
deteriorating effects. In many of these situations it would be desirable 
to be able to replace the bone structure or strengthen the bone structure. 
In providing materials to substitute for natural bone, teeth, or other 
calcified tissues, there are a number of restraints on the natural 
composition of the material. 
Dental applications might prefer a fluoride substituted hydroxyapatite, 
i.e. francolite, that would reduce solubility and increase resistance to 
decay. 
The material should ideally possess certain characteristics that facilitate 
the production, storage life, and biomedical application of the material. 
Specifically, a material which could be a material which could be 
fingerpacked in an open surgical procedure or percutaneously injected as a 
flowable composition to fill voids or completely fill-in areas deficient 
of hard bone is very desirable. Where the material is to be placed in the 
body and formed and hardened in situ, a variety of considerations come to 
the fore. For example, the rate at which hydroxyapatite forms as well as 
the extent to which the formation of hydroxyapatite is exothermic or may 
generate gas can also be important. Where the reaction is highly 
exothermic, it may cause thermal necrosis of the surrounding tissue. 
As the final form of the material must be stable under physiological 
conditions, so must the form in which the material is introduced be stable 
while it is hardening in the environment to which it is introduced, as 
must be any intermediate products of the formation reaction. 
The material should also be physiologically acceptable at all phases of 
curing to the final product, so as to avoid the initiation of clotting, 
inflammatory responses, and the like. Two different forms of apatite are 
particularly desirable: One being an hydroxyapatite or a fluoridated 
derivative thereof that is non-resorbable in vivo; the other includes 
forms of apatite that are substantially resorbable in vivo. In addition, 
both forms of apatite must usually be strong and non-friable. Furthermore, 
there should be a strong adhesion between the material and the remaining 
bone or calcified tissue. Also, the material should desirably be able to 
substitute some of the other functions of natural bone such as: 
accommodating stem cells; allowing infiltration by cells normally resident 
in natural bone such as osteoclasts, osteoblasts, and the like; allowing 
remodeling of the material by the infiltrating cells followed by new bone 
in-growth; and acting in metabolic calcium exchange in a manner similar to 
native bone. 
Carbonate has been shown to inhibit crystal growth of HAp (Blumenthal, et 
al., Calcif. Tissue Int. (1984) 36:439-441; LeGeros, et al., "Phosphate 
Minerals in human tissues", in Phosphate Minerals (Berlin), J. Nriagu 
(eds): Springer, 1984, pp. 351-385; LeGeros, et al., J. Dent. Res. (1989) 
68:1003; Nauman and Neuman, The Chemical Dynamics of Bone Mineral, 
University of Chicago Press, Chicago, (1958); Newesley, Arch. Oral Biol. 
(1961) 6:174-180; Posner, Clin. Orthop. (1985) 200:87-99). Carbonates are 
present in the apatites of hard tissues, and their presence alters the 
properties of stoichiometric apatite. Carbonate has been described as 
causing: 1) a reduction in crystallite size, 2) changes in the 
morphologies of the mineral phase from needles and rods to equi-axis 
crystals (spheroids), 3) contraction of the a-axis, as well as an 
expansion in the c-axis, 4) internal strain, and 5) chemical instability 
(LeGeros, et al., supra, 1984; LeGeros, et al., supra, 1989). All of these 
factors lead to higher solubilities of carbonate-substituted HAp. The 
x-ray diffraction patterns as well as the radial distribution function are 
changed considerably in that as the concentration of carbonate increases, 
the patterns become more amorphous in character (LeGeros et al., supra 
1989; Glimcher, M. J., "Recent studies of the mineral phase in bone and 
its possible linkage to the organic matrix by protein-bound phosphate 
bonds", Phil. Trans. R. Soc. London Ser. B., 304, 479-508). The line 
broadening observed in the diffraction pattern is caused by decreasing 
crystallite size and crystallinity. In addition to inhibiting HAp crystal 
growth, carbonate substitution markedly increases the solubility of HAp 
(Nelson, et al., Ultramicroscopy (1986) 19:253-266. Another interesting 
experimental finding was that, whether the carbonates are structurally 
bound within, or absorbed onto HAp, differences in dissolution behavior 
were observed. This suggests that dissolution increased in HAps containing 
structurally bound carbonates, while decreasing in HAps with absorbed 
CO.sub.3.sup.2-. The decrease in dissolution was explained by the fact 
that hydronium ions had to compete for the surface of HAp, hence the 
deposition of the CO.sub.3.sup.2- layer was required. 
The extent of carbonate uptake during HAp precipitation under normal 
physiologic conditions is approximately 1% by weight CO.sub.3.sup.2- 
(Posner, supra, 1985). Bone consists of approximately 4% by weight 
CO.sub.3.sup.2-. Thus, HAp precipitation reactions in air generally 
contain relatively low concentrations of carbonate. Bone mineral apatite 
with a level of carbonate between 2% and 10% by weight has been referred 
to by convention as dahllite (McConnell, J. Dent. Res. (1952) 31:53-63 and 
McConnell, Clin. Orthopaed. (1962) 23:253-268). 
Carbonates can substitute in both the Z and Y sites of the apatite 
structure, and it is generally accepted that carbonates substitute for 
PO.sub.4.sup.3- groups during precipitation reactions leading to HAp 
formation. More specifically, HAp products formed at lower temperature 
exhibit carbonate substitution at the phosphate sites, and due to its 
smaller size, a decrease in the a-axis of the apatite results (LeGeros, et 
al., supra, 1984 and LeGeros, et al., supra, 1989). Conversely, in most 
high temperature apatites, the carbonates are found in the vicinity of the 
six fold axis, where they replace hydroxyl ions. Since the carbonate is 
larger than the hydroxl ion, an increase in the a-axis results (Brown and 
Chow, (1986), supra). 
The skeleton is the reservoir for almost all of the body's calcium and most 
of its phosphorus and magnesium (Avioli and Krane, Metabolic Bone Disease 
and Clinically Related Disorders, 2nd Ed., 1990, W. B. Saunders Co., 
Philadelphia, p. 2). The carbonate levels in human enamel have been shown 
to increase in concentration from the surface to the dentin. The carbonate 
concentration in the surface enamel has also been shown to decrease with 
age (Brudevold and Soremark, Chemistry of the mineral phase of enamel, 
Miles (ed.), In: Structural and Chemical Organizations of Teeth. Academic 
Press, New York, 1967, Vol. II, p. 247. The ease of ionic substitution in 
the lattice of apatite allows for the ionic substitution of ions from the 
fluids surrounding the bone, and vice versa. This implies that hard 
tissues act as a regulatory reservoir for certain ions by incorporating 
ions into its structure when ionic concentration in the serum rises too 
high, and dissolving ions when the body is deficient in them. Possible 
candidates for this form of regulation might include some of the inorganic 
constituents of serum such as ionized and complexed calcium, inorganic 
phosphates, magnesium, bicarbonate, sodium, chloride, potassium, among 
others (Eidleman, et al., Calcif. Tissue Int. (1987a) 41:18-26; Eidelman, 
et al., Calcif. Tissue Int. (1987b) 40:71-78; Meyer and Fleisch, Miner. 
Electrolyte Metab. (1984) 10:249-258). 
Carbonate is especially important in hard tissue in that it apears to be 
required for the cellular infiltration of bone by osteoclasts, osteoblasts 
and other bone resident cells. Since osteoclasts, osteoblasts and the like 
are involved in mineral replacement and bone remodeling, any synthetic 
apatite-associated bioimplant would preferably use a carbonated form of 
apatite, or dahllite. Because dahllite can be remodeled by the bodies 
natural processes, the dahllite component of an implant should, through 
the action of osteoclasts and osteoblasts, eventually be replaced by 
natural bone. Thus, dahllite implants should eventually gain many or all 
of the desirable features of natural bone such as increased strength, 
elasticity and durability. 
Previous methods of chemically forming monolithic bodies of hydroxyapatite 
have not produced dahllite or hydroxyapatites with physiologically 
significant levels of structurally incorporated carbonate. This is 
primarily because the acid present in the reactions of other methods tend 
to react with the carbonate to produce gaseous CO.sub.2. Gaseous escape 
removes carbonate from the reaction which is forming apatite and can 
result in a product that is substantially more friable than that generally 
desired by virtue of the trapped gas bubbles disrupting the structural 
integrity of the product. Thus, a major obstacle to the production of 
dahllite has been devising a method to maintain carbonate in the product 
despite the presence of the acid required to form the apatitic structure. 
Relevant Literature 
Patents of interest include U.S. Pat. Nos. 3,787,900; 3,913,229; 3,679,360; 
4,097,935; 4,481,175; 4,503,157; 4,612,053; 4,659,617; and 4,693,986. See 
also, Arends and Jongebloed, Rec. Trav. Chim. Pays-Bas (1981) 100:3-9. Use 
of calcium phosphate as a sealer-filler material is described in Chohayeb 
et al., J. Endodontics (1987) 13:384-387 and Lowenstam and Weiner, On 
Biomineralization, (1989), Oxford University Press, New York. See also, 
Ohwaki et al., 13th Ann. Mtg. of the Soc. for Biomaterials, Jun. 2-6, 
1987, New York, N.Y., p209. 
SUMMARY OF THE INVENTION 
Compositions comprised of dahllite, analogs thereof, or otherwise 
carbonate-substituted forms of hydroxyapatite (dahllite-like compositions) 
are provided that are useful in a variety of biomedical applications. The 
compositions can be prepared such that they are flowable, moldable, and 
capable of hardening in situ. The compositions harden into monolithic 
polycrystalline structures that can be shaped subsequent to hardening. 
DESCRIPTION OF THE SPECIFIC EMBODIMENTS 
Compositions are provided that are comprised of substantially pure (greater 
than 80% by weight) dahllite-like compositions referred to as carbonated 
hydroxyapatite that can be produced substantially free of the blood-borne 
or organic components of natural bone. The compositions can be used to 
substitute many of the functions of naturally occurring calcified tissues 
or in the repair of such tissues, in particular teeth and bone. The 
dahllite or francolite-like products can be readily formed by combining 
the wet and dry reactants to provide a substantially uniform mixture, 
shaping the mixture as appropriate, and allowing the mixture to harden. 
During hardening, the mixture crystallizes into a solid and essentially 
monolithic apatitic structure. The dahllite and francolite-like apatitic 
compositions can also be shaped subsequent to hardening. Alternatively, 
the dahllite or francolite-like apatitic compositions can be precursor 
reaction mixtures placed into an appropriate section of the body and 
hardened and/or shaped in situ. 
The composition of the carbonated hydroxyapatite may vary, frequently being 
non-stoichiometric in having incorporated extra hydrogen atoms. Also, the 
calcium/phosphate ratio may vary, where the ratio may be as low as 1.33 
(1.67 is the natural ratio), so that there is a defective lattice 
structure from the calcium vacancies and as high as 2.0. For a ratio of 
1.33, there will be two calcium ions absent. The extra hydrogens may be up 
to about 2 hydrogen ions per phosphate, usually not more than about one 
hydrogen ion per phosphate. The ions will be uniformly distributed 
throughout the product and for the most part, the composition will be 
monophasic having a single crystal structure. As compared to sintered 
hydroxyapatite and hydrothermally prepared hydroxyapatite, the X-ray 
diffraction and Fourier transform infra-red spectra of the subject 
compositions are substantially different. 
The reactants will generally consist of a phosphoric acid source 
substantially free of unbound water, an alkali earth metal, particularly 
calcium, source, optionally crystalline nuclei, particularly 
hydroxyapatite or calcium phosphate crystals, calcium carbonate, and a 
physiologically acceptable lubricant, such as water, which may have 
various solutes. The dry ingredients may be pre-prepared as a mixture and 
subsequently combined with the liquid ingredients under conditions where 
substantially uniform mixing occurs. Where gases are evolved the mixture 
is agitated to cause the release of large pockets of gas. 
The phosphoric acid source may be any partially neutralized phosphoric 
acid, particularly up to and including complete neutralization of the 
first proton as in calcium phosphate monobasic. Alternatively or 
additionally, it can consist of orthophosphoric acid, possibly in a 
crystalline form, that is substantially free of uncombined water. The acid 
source will generally be about 15 to 35 weight percent of the dry 
components of the mixture, more usually 15 to 25 weight percent. 
In selecting the calcium source, particularly where the calcium source 
serves a dual role of providing calcium and acts in a neutralizing 
capacity, one must consider that the desired final product will depend on 
the relative ratios of calcium and phosphate. Calcium sources will 
generally include counterions such as carbonate, phosphate or the like. Of 
particular interest are dual sources of calcium phosphate and phosphate 
such as tetracalcium phosphate (C.sub.4 P) or tricalcium phosphate 
(C.sub.3 P). Tetracalcium phosphate or tricalcium phosphate may typically 
be present in the mixture at from about 0 to 70 weight percent, more 
usually from about 0 to 40 weight percent, and preferably from about 2 to 
18 weight percent of dry weight of the dry components of the mixture. With 
calcium carbonate present to neutralize the acid and to serve as a source 
of calcium and carbonate, the reaction will result in relatively little 
temperature rise; however, there is substantial evolution of gas which 
must be released during mixing. Calcium carbonate will be present in the 
mixture from about 2 to 70 weight percent, more usually from about 2 to 40 
weight percent, and preferably from about 2 to 18 weight percent of dry 
weight of the dry components of the mixture Calcium hydroxide may also be 
present in the mixture from about 0 to 40 wt. %., more usually from about 
2 to 25 wt. %, and optimally from about 2 to 20 wt. %. 
Halides such as fluorine and chlorine may be added to form fluorapatite 
(francolite), or chlorapatite respectively. Various sources of fluoride or 
chloride may be employed. Generally, the sources will include either 
soluble salts such as calcium chloride, calcium hexafluorosilicate or 
sodium fluoride or, less desirably, the source may be added as a dilute 
acid in the aqueous lubricant, generally at concentrations of less than 
about 1M. Halides, if present at all, will constitute from about 0 to 4 
weight percent, more usually from about 2 to 4 weight percent, preferably 
from about 3 to 4 weight percent of dry weight. Usually at least about 5, 
more usually at least about 10% of the hydroxyl groups will be replaced, 
and up to 100%. Francolite is of particular interest because of the 
potential dental applications of this partially fluorine substituted form 
of dahllite. 
The various dry components may be combined prior to the addition of the wet 
components. Mixing will be used to combine the ingredients and can be used 
to regulate the extent of the inter-ingredient reactions. Any or all of 
the dry ingredients may be added prior to the initiation of mixing or 
prior to the completion of mechanical mixing. Methods of mixing can 
include ball milling, Brabender mixing, rolling between one or more 
rollers and a flexible container, or the like. Preferably, mixing will be 
thorough and will occur for a relatively short time or until a uniform 
dispersal of ingredients is obtained. 
By varying the proportion of liquid lubricant, particularly water, added to 
the subject mixtures, the fluidity of the composition can be varied with 
respect to flowability and viscosity. Besides or in combination with 
water, other water miscible pharamacologically acceptable liquids may be 
used, particularly alkanols, more particularly polyols, such as ethylene 
glycol, propylene glycol or glycerol, usually being present in less than 
about 10 volume percent in an appropriate medium. The liquid will 
generally be from about 15 to 50, more usually from about 20 to 35 weight 
percent of the entire composition. Various solutes may be included in the 
aqueous medium. Of particular interest is the use of a gel or colloid, 
which has as a solute alkali metal hydroxide, acetate, phosphate, or 
carbonate, particularly sodium, more particularly phosphate or carbonate, 
at a concentration in the range of about 0.01 to 2 m, particularly 0.05 to 
0.5 m, and at a pH in the range of about 6-11, more usually about 7-9, 
particularly 7-7.5. 
Implantation may be by syringe or catheter injection; particularly, the 
composition may be used as a paste that passes through a needle in the 
range of about 10-18 gauge, preferably about 14-16 gauge. Alternatively, 
if less lubricant is added, the composition is kneadable or moldable, 
being capable of forming clay-like putty that may be molded prior to 
setting. By varying the amount of lubricant employed, the setting time of 
the compositions can also be varied. 
After mixing, the mixture is allowed to anneal while remaining quiescent, 
followed by an extended period of time during which the mixture hardens. 
During hardening, crystal growth occurs and the product becomes an 
integral mass. Hardening will take at least about 5 minutes, usually at 
least about 15 minutes, and not more than about 20 minutes. Compounds 
produced in this manner will have a wide variety of desirable properties 
for use in physiological applications. 
The claimed compositions will contain, structurally incorporated into the 
apatitic structure, between about 2% and about 10% carbonate by weight, 
usually between 2.5% to 7%, and optimally between about 4% to about 6% 
carbonate by weight. 
The subject compositions are biocompatible having a pH in the range of 
about 5.5-8.5, usually in the range of about 6-7.5. They can be prepared 
so that they can be administered to an environment having a temperature in 
the range of about 0-45.degree. C., usually 20-40.degree. C., and 
optimally about normal physiological temperature, 37.degree. C. The 
compositions have low or no toxicity when prepared in accordance with 
described methods, are substantially inactive as to detrimental 
interactions with various host components in vivo, and are readily 
implantible. Furthermore, they are readily resorbable in vivo so as to be 
replaced by natural bone. 
Various additional components may be included during the formation of the 
carbonated hydroxyapatite, dahllite. Of particular interest are 
pharmacologically active agents, proteins, polysaccharides, or other 
biocompatible polymers, or the like. Of particular interest are proteins 
involved in skeletal structure such as different forms of collagen, 
especially Type I, fibrin, fibrinogen, keratin, tubulin, elastin, and the 
like, or structural polysaccharides, such as chitin. Pharmacologically 
active agents might include drugs that enhance bone growth, serve as a 
variety of cell growth factors, or act as anti-inflammatory or 
anti-microbial agents. Examples of such proteins might include but not be 
limited to: bone morphogenetic protein, cartilage induction factor, 
platelet derived growth factor, and skeletal growth factor. 
Pharmacologically active or structural proteins may be added as an aqueous 
dispersion or solution. Usually the protein will be present in from about 
1-10 wt % of the aqueous dispersion. The protein will be present in the 
final composition after setting in from about 0.01 to 10, usually from 
about 0.05 to 5 weight percent. The amount of water added to the 
compositions to which protein in aqueous dispersion has also been added 
will be adjusted accordingly. By varying the proportions of the reactants, 
compositions with varying and predictable rates of resorption in vivo, can 
be made. Thus, the subject compositions enable one of ordinary skill in 
the art to add drug and inorganic components both subsequent to and 
during, and possibly prior to, the formation of the subject compositions 
in order to practice an implantible or injectable time-release delivery 
platform for drugs, inorganic mineral supplements, or the like. 
When used as cements or fillers, the subject compositions bond to other 
apatites when applied to an apatitic surface, such as bones or teeth which 
are mainly comprised of dahllite and collagen. The applicable compositions 
are able to strongly adhere and bond to surfaces that are wet or coated 
with saliva, blood or lymphatic fluid, will fill voids, and conform to 
irregular surfaces such as concavities and convexities. The compositions 
may be applied as a continuous mass without the formation of fragments or 
loose particles to a significant degree. Furthermore, the subject 
compositions are found to be structurally compatible in providing for the 
structural functions of replaced connective tissue. 
The subject compositions can be used to form carbonated hydroxyapatite 
coatings on bioimplants or other formed objects. 
The subject composition, as a flowable or formable product, can serve as a 
bone cement, or an infiltrate cement for the treatment of osteoporotic 
bone. 
Paste or clay-like mixtures of product are provided that may be formed and 
hardened into a monolithic carbonated hydroxyapatite product, either 
externally or in situ. 
Of particular interest is preparation of the subject carbonated 
hydroxyapatite by a process whereby a calcium source, at least one 
component of which is calcium carbonate, and an acidic phosphate source, 
optionally comprised of ortho phosphoric acid crystals substantially free 
of uncombined water, are mechanically mixed for sufficient time for a 
partial reaction of said calcium source and acidic phosphate source. The 
partially reacted composition can be subsequently mixed with a 
physiologically suitable lubricant which varies the fluidity of the 
product, allows the substantially complete reaction of the reactants, and 
eventually results in a monolithic solid carbonated hydroxyapatite 
product. The final mixture may be subsequently shaped and hardened, 
hardened then shaped, or placed in the body and hardened in situ. The 
carbonated hydroxyapatite of the subject process will have substantially 
reduced or non-exothermic setting which may better provide for the 
stability of introduced pharmacological agents, and, when hardened in 
situ, is desirable for purposes of patient comfort. The compositions of 
this process are also applicable as bone cements or fillers, dental or 
endodontic filling agents, coatings for bioimplantible substrates, or 
formed into suitable shapes before or after hardening into a monolithic 
structure. 
The calcium source used in the above process will typically include a 
mixture of tetracalcium phosphate (C.sub.4 P) and calcium carbonate (CC) 
with C.sub.4 P typically present in from about 55 to 75 wt. %, or more 
usually 60-70 wt. %, and CC typically present in from about 1 to 40 wt. %, 
or more typically 2 to 18 wt. % of the dry weight of the total reaction 
mixture. 
The acid phosphate source will be about 15 to 35, or more usually 15 to 25 
wt. % of the dry weight of the reaction mixture. 
An alternative formula will typically include a mixture of tricalcium 
phosphate (C.sub.3 P), calcium carbonate (CC), and calcium hydroxide (CH) 
with C.sub.3 P typically present in from about 50 to 90 wt. %, or more 
usually 75 to 90 wt. %, CC typically present from about 1 to 40 wt. % or 
more usually 2 to 18 wt. %, and CH typically present from about 0 to 40 
wt. % or more usually 2 to 20 wt. % of the dry weight of the total 
reaction mixture. 
The acid phosphate source for this alternative mixture will be about 5 to 
35 wt. % or more usually 5 to 25 wt. % of the dry weight of the reaction 
mixture. 
A fluoride source may generally be added to the mixture and, if at all 
present, will be in an amount from about 0 to 4 wt. %, preferably 3 to 4 
wt. % of dry weight. 
After the dry ingredients are combined, the reactants will be placed in 
intimate contact by mechanical mixing or milling. Prior to the completion 
of mixing/milling, proteins and/or small organic molecules, especially 
those containing pharmacological significance as indicated earlier, may be 
added to the mixture to alter the physical or physiological properties of 
the final product. The amount of additive will generally vary from about 1 
to 40 weight percent, more usually from about 1 to 25 weight percent of 
the inorganic materials. It may be preferred that the additive be combined 
with the inorganic materials before mixing/milling. 
Mechanical mixing may be by any form that results in an intimate mixing of 
the reactants. A variety of equipment may be used for these purposes 
including ball mills, planetary mills, centrifugal mills, mechanofusion 
systems, air pulverizers, jet mills, vibratory mills, colloid mills, 
attrition mills, disc mills, and the like. 
The course of mixing can be monitored by periodically removing samples and 
testing whether or not the samples result in the formation of a product 
with the desired properties after mixing with as aqueous medium and 
subsequent hardening. 
During mixing or milling, the walls of the mixing vessel may be 
periodically scraped to better promote a more uniform product. The milling 
media should remain stable as inert throughout the process as would media 
such as alumina, zirconia, tungsten carbide, boron carbide, etc. 
The product of the above process will have undergone a relatively stable 
partial reaction and will require less lubricant to provide a workable 
mixture as well as a reduced setting time. 
All of the above-mentioned products or their precursors may be sterilized 
by gamma-irradiation or other applicable methodologies prior to 
bioimplantation. 
The following example is offered by way of illustration and not by way of 
limitation. 
EXPERIMENTAL

EXAMPLE 1 
In a chilled mortar, 11.54 g of tetracalcium phosphate (TCP), 1.40 g of 
calcium carbonate, 2.06 g of ortho-phosphoric acid and 7.5 g of 0.1 m 
sodium phosphate were mixed with a chilled pestle (mixed bases for 15 
sec., followed by 30 sec. of mixing of both acid and bases, then 3 min. of 
wet mixing of the combined solution, acids and bases). The completed mixes 
were injectable and free flowing and were immediately put into bovine 
serum with (0.1% sodium azide) and cured at 37.degree. C. for about 2 
weeks. At the end of the 2 week cure, the samples were rinsed with 
deionized water, frozen in liquid nitrogen, then lyophilized overnight. 
Sample aliquots were analyzed by Fourier transform infrared spectroscopic 
analysis (FTIR) using pressed KBr pellets and by carbon coulometry using 
acidification for total inorganic carbon analysis and by combustion for 
total carbon analysis. The samples were further assayed for carbonate 
content in duplicate. The results of these assays are presented in Table 1 
which shows that the subject compositions reacted to form carbonated 
hydroxyapatite having a dahllite crystal structure as confirmed by the 
weight percentage of carbonate contained in the resulting apatites. 
TABLE 1 
______________________________________ 
Formulation Mineralogy 
% Carbonate 
______________________________________ 
A 11.54 g C.sub.4 P(c), 1.4 g 
98% Dahllite 
4.53 
CC, 2.06 g oPA, 7.5 g 
0.1 m SP 
B 11.54 g C.sub.4 P(f), 1.4 g 
98% Dahllite 
4.83 
CC, 2.06 g oPA, 7.5 g 
0.1 m SP 
______________________________________ 
Abbreviations: 
C.sub.4 P = Tetracalcium phosphate, fine (f) 3 microns, course (c) 10 
microns. 
CC = Calcium carbonate. 
oPA = Orthophosphoric acid. 
SP = Sodium phosphate, dibasic, 7hydrate. 
EXAMPLE 2 
A number of dry formulations were prepared having the compositions as set 
forth in Table 2. 
TABLE 2 
______________________________________ 
Ingredient 1 2 3 
______________________________________ 
Tetracalcium phosphate 
45.0 43.95 13.73 
Calcium oxides 0.17 0.33 0.56 
Calcium carbonate 
5.85 11.4 4.0 
Ca(H.sub.2 PO.sub.4).sub.2.H.sub.2 O 
15.5 
Orthophosphoric acid 11.76 4.41 
Na.sub.2 SiF.sub.6 
0.29 0.25 0.09 
______________________________________ 
Each of the above formulations were ball milled in an alumina/silica mill 
jar with 0.5".times.0.5" alumina cylinders, where the container was from 
25 to 50 volume percent full. The milling usually was continued for about 
16 hours. In many cases, some caking was observed, particularly at the top 
of the container. The gasket at the top of the container to enclose the 
cover was cut, so as to allow for the release of gas. The mixing was at 
about 50 rpm. 
After completion of the milling, the composition was combined with water, 
generally using about 0.35 parts of water to 1 part of solids. For 
preparing samples, 5 g of the solid mixture was combined with 1.75 g of 
deionized water and the mixture kneaded for about 5 min. The composition 
was introduced into a mold, allowed to set, and the sample removed for 
compressive strength testing. In some instances, the samples could not be 
easily removed from the mold, which could have affected the observed 
compression properties. The following table indicates the results, where 
the results are reported as the average of from 3 to 4 determinations on 
different samples from the same composition. 
TABLE 3 
______________________________________ 
Compressive 
Ex. Weight (g) Load (lbs.) 
Strength (psi) 
______________________________________ 
1 0.72 367.9 8389 
______________________________________ 
Considerable variation was noticed in the results. In example 1, the 
variation was from 5620 to 11794. Thus, while samples can be obtained 
having compressive strengths in substantial excess of 10,000 psi, the 
reasons why other samples from the same composition do not provide the 
same properties is believed to be related to defects in the specimen 
related to sample preparation. However, in any sample, products having 
properties in substantial excess of 10,000 psi compressive strength are 
achievable. 
It is evident from the above results that the subject composition and the 
products derived therefrom provide a unique alternative to standard 
hydroxyapatite materials. Unlike standard hydroxyapatite compositions, the 
subject dahllite compositions can be constructed to contain carbonate at 
levels near or exceeding those normally occurring in natural bone. Since 
carbonate is intimately involved in the processes by which normal 
bone-resident cells are able to infiltrate, resorb and replace natural 
bone, the subject compositions provide products that better mimic natural 
calcified tissues in both form and function. 
All publications and patent applications mentioned in this specification 
are indicative of the level of skill in the art to which this invention 
pertains. All publications and patent applications are herein incorporated 
by reference to the same extent as if each individual publication or 
patent application was specifically and individually indicated to be 
incorporated by reference. 
Although the foregoing invention has been described in some detail by way 
of illustration and example for purposes of clarity of understanding, it 
will be obvious that certain changes and modifications may be practiced 
within the scope of the appended claims.