Patent 6,972,130

The present invention provides a synthetic, poorly crystalline apatite (PCA) calcium phosphate containing a biologically active agent and/or cells (preferably tissue-forming or tissue-degrading cells). The compositions provided by the present invention are useful for a variety of in vivo and in vitro applications, including drug delivery (for example, to bony sites, the central nervous system, intramuscular sites, subcutaneous sites, interperitoneal sites, and occular sites) tissue growth (preferably bone or cartilage) osseous augmentation, and methods of diagnosing disease states by assaying tissue forming potential of cells isolated from a host. The invention also provides methods of preparing delivery vehicles, of altering delivery vehicle characteristics, and of delivering biologically active agents to a site. The invention further provides in vitro cell culture systems and cell encapsulation materials. The invention is useful for both medical and veterinary applications.

SUMMARY OF THE INVENTION

The present invention provides a synthetic, poorly crystalline apatitic calcium phosphate material that has excellent biocompatibility, resorbability, and processability characteristics and is useful in drug delivery and cell seeding (in vivo and in vitro) applications.

The synthetic PCA material utilized in the present invention is compatible with cells and with a wide array of biologically active agents. The material can be employed to deliver agents or cells to any of a variety of sites in the body, or can be used in vitro. The material is characterized by a distinctive X-ray diffraction pattern that reveals its poor crystallinity. Preferable, the material has a calcium to phosphate ratio in the range of about 1.1 to 1.9. More preferably, this ratio is in the range of about 1.3 to 1.5.

The PCA material utilized in the present invention is strongly bioresorbable. That is, when an implant comprising at least 1 g of material is implanted in pellet form in an intramuscular or subcutaneous site, at least approximately 80%, preferably 90-95%, and most preferably >95%, of the material is resorbed within one year, preferably within 9 months, 6 months, 3 months, and, ideally 1 month. More preferably, at least 80%, preferably 90-95%, and most preferably 22 95%, of a 5 g implant is resorbed within these time frames. It will be appreciated that the conformation of the material (e.g., in a sphere is compared with a rod or other shape) may affect is resorption rate. Furthermore, the resorption rate of the delivery vehicle can be varied through its manner of preparation.

In preferred embodiments of the present invention, the synthetic PCA material is formed in a reaction in which at least one amorphous calcium phosphate (ACP) precursor is exposed to a promoter. In particularly preferred embodiments, the promoter comprises a second calcium phosphate material. The reaction conditions employed to produce the PCA material utilized in the present invention are mild, so that biological agents or cells can be incorporated into the material during the formation reaction, if desired. Alternatively, the agents may be incorporated after the delivery vehicle is made. The delivery vehicle material may be formed into any of a variety of useful delivery shapes, either before or after the introduction of biologically active agent or cell, and may be delivered to the site by, for example, injection or surgical implantation. The material may be introduced into a site in a wet, non-hardened state (i.e., as a hydrated precursor) and allowed to harden in situ. The vehicle may alternately be hardened in vitro at an elevated temperature, generally at or above 37° C., and thereafter surgically implanted into a subject (animal or human).

The PCA material of the present invention may be fabricated in vitro either in the presence or absence of the biologically active agent or cell. Alternatively the biologically active agent or cell may be added post-hardening by exposing the pre-formed vehicle to the agent.

The present invention therefore provides vehicles for delivering biologically active agents, which vehicles comprise to PCA calcium phosphate and a biologically active agent. The inventive vehicles optionally comprise, for example, other bioresorbable materials, erosion rate modifiers, cells, or other factors that modify one or more characteristics of the vehicle (such as its strength, adherence, injectability, frictional characteristics, etc.). One advantage of the delivery system of the present invention is that it allows a high local concentration of drug to be achieved, which is particularly useful with drugs that have toxic side effects and also with labile drugs.

The invention also provides methods of preparing delivery vehicles, of altering delivery vehicle characteristics, and of delivering biologically active agents to a site. Preferred delivery sites include both in vitro and in vivo sites. The delivery vehicles of the invention are suitable for delivery into human or animal sites. Preferred in vivo sites include bony sites, intramuscular sites, interperitoneal sites, subcutaneous sites, central nervours system sites, and occular sites.

The present invention additionally provides therapeutic, structural, or cosmetic implants comprising the inventive PCA material and at least one cell. Preferably, the at least one cell is a bone-forming or bone-degrading cell. Particularly useful cells types include chondrocytes, osteocytes, osteoblasts, osteoclasts, mesenchymal stem cells, fibroblasts, muscle cells, hepatocytes, parenchymal cells, cells of intestinal origin, nerve cells, and skin cells, and may be provided as primary tissue explants, preparations of primary tissue explants, isolated cells, cell lines, transformed cell lines, and host cells. The implants may also comprise additional components such as biologically active agents or factors that alter the characteristics (such as resorbability, strength, adherence, injectability, frictional characteristics, etc.).

The invention also provides methods of preparing such implants; methods of growing bone or cartilage in vivo or in vitro, a natural sites or ectopic sites; methods of osseous augmentation; and methods of diagnosing disease states by assaying tissue-forming potential of cells isolated from a host. The invention also provides in vitro cell culture systems and cell encapsulation matrices.

DESCRIPTION OF PREFERRED EMBODIMENT

The PCA Material

The PCA material of the present invention is described in co-pending applications U.S. Ser. No. 08/650,764 and/or U.S. Ser. No. 08/446,182, each of which is incorporated herein by reference. The material is also described in a set of related applications, entitled "Delivery Vehicle"; "Conversion of Amorphous Phosphate to Form a Novel Bioceramic"; "Orthopedic and Dental Ceramic Implants"; and "Bioactive Ceramic Composites", each of which is on even date herewith and is incorporated herein by reference. In light of the bread of disclosures in each of these related applications, the details of the inventive PCA materials will not be belabored here. A summary of its characteristics will suffice.

The PCA material employed in the present invention is characterized by its biocompatibility, its biological resorbability and its minimal crystallinity. The material may be highly porous and rapidly resorbable or of decreased porosity and slowly resorbable. Its crystalline character is substantially the same as natural bone, and lacks the higher degree of crystallinity seen in the bone substitute known to the art. The inventive PCA material also is biocompatible and not detrimental to the host.

The PCA material of the present invention may be implanted in a patient in a paste or putty form (i.e., as a hydrated precursor). Since the inventive reaction that produces the hardened PCA material can be initiated outside the body, and proceeds slowly at room temperature, the possibility that the material will "set up" prior to application to the surgical site and become unusable is minimized. The reaction accelerates significantly at body temperature and the material hardens in place. This feature is particularly useful in the surgical setting, where custom fitting of the device to the implant location is typically required. For example, in some preferred embodiments of the invention, an antibiotic and/or regenerative factor is delivered to a fracture site. In such embodiments, the inventive paste containing the therapeutic agent will be applied to and used to fill a fracture site, as well as to deliver the desired agent.

Alternatively, the inventive PCA material may be pre-hardened outside the body, loaded with the desired biological agent or cell(s), and implanted at a later time. This approach is useful in those situations where custom shapes are not essential, and where production of large numbers of implants is desired.

Generally, the formation reaction of the present invention is completed after application to the surgical site. The material typically hardens in less than five hours, and substantially hardens in about one to five hours, under physiological conditions. Preferably, the material is substantially hardened within about 10-30 minutes. The consistency and formability of the PCA material, as well as the speed of the formation reaction, may be varied according to the therapeutic need by modifying a few simple parameters.

The resorbability of the PCA material employed in the instant invention is attributable to the combination of its porosity, its chemical composition, and its crystallinity. A patites have reduced crystalline characters and display somewhat increased solubility in aqueous solution systems when compared with more crystalline species. The low crystallinity of the inventive PCA material, and/or the presence of stable amorphous domains within it, is believed to promote its resorbability in biological systems.

The resorbability of the PCA material of the present invention can be modified by altering its density and/or porosity. Porosity facilitates both the diffusion of substances to and from the interior of the material and, in certain applications, the penetration of cells and cell processes into the material matrix. Drug delivery materials of lower porosity tend to resorb more slowly in vivo than do those of higher porosity. In one embodiment of the invention, porosity is increased through the use of a dry mixture of controlled particle size reactants; in other embodiments, chemical or physical etching and leaching techniques are employed.

Thus, different embodiments of the present invention provide PCA materials with different resorption rates. Selection of reactants, porosity, final crystallinity, and amounts and types of crystallization inhibitors employed yields difficulty embodiments of the PCA material of the present invention, so that, in different embodiments, 1 g of material is resorbed (i.e., at least 80%, preferably 90-95%, and most preferably >95%, resorbed) within any desired time period from 2 weeks to 1, 3, 6, and 9 months, to 1 year.

In a preferred embodiment of the present invention, the reaction that produces the PCA material is initiated by adding physiological saline to a mixture of two dry components so that a thick paste forms that hardens in about a half an hour. Other aqueous agents, such as serum, tissue culture medium, or another buffered solution or distilled water, may be used in place of saline. Most often, the resulting resorbable PCA material will be "calcium deficient", with a calcium to phosphate ratio of less than 1.5 as compared to the ideal stoichiometric value of approximately 1.67 for hydroxyapatite.

The invention provides a test for identifying suitable PCA materials and reactive precursors. Specifically, precursors are combined, are hydrated with a limited amount of water (so that a paste or putty is formed), and are allowed to harden into a PCA material. Desirable precursors are capable of hardening in a moist environment, at or around body temperature. The hardened product is then placed intramuscularly or subcutaneously in a test animal. Desirable materials are those that, when implanted as an at least 1 g pellet are at least 80%, preferably 90-95%, and most preferably >95%, resorbed within 1 year (or less). Preferably, the material can be fully resorbed. Generally, it is easier to test resorption of gram quantities of material in subcutaneous sites.

The PCA material of the present invention is formed in a reaction that employs at least one amorphous calcium phosphate (ACP) precursor, preferably an activated ACP (see, for example, Examples 1-4). In some instances, the reaction may employ only one precursor ACP, which is converted in a controlled fashion in part or whole to the PCA material of the invention. Alternatively, the reaction may employ a promoter that comprises one or more additional precursors (preferably one or more calcium and/or a phosphate sources), that combine with the ACP to yield the PCA material of the invention. Also, a non-participating promoter may be employed to facilitate conversion of the activated ACP to the inventive PCA material. In any event, reaction that can be initiated outside the body, that can be carried on in a paste-like configuration, and that significantly accelerate at 37° c. leading to a hardened calcium phosphate product are greatly preferred.

The conversion of ACP to a PCA material is promoted in the presence of water. Generally, the ACP is provided as a powder is combined with any other reactants (e.g. a second calcium phosphate), and is exposed to a limited amount of water, so that a paste or putty is formed. The hydrated precursor then hardens, and the hardening is associated with formation of the PCA material. It is an aim of this invention to provide methods which promote the conversion of ACP to a PCA material in a controlled fashion, producing a hydrated precursor paste or putty that hardens predictably and has utility in dental, orthopedic, drug delivery, cell therapy, and/or other applications. The promoters used to accomplish this conversion may themselves be converted to PCA material, or may participate in other chemical or physical reactions. Some preferred promoters may also remain unchanged during the conversion, providing a catalytic or nucleator function. Particularly suitable in this regard are substances that provide reactive surfaces that weakly promote crystallization to produce PCA calcium phosphate.

ACP precursors only: When amorphous calcium phosphate is used as the sole precursor to produce a reasonable PCA material, it is important to control the neutral tendency of the ACP to convert to highly crystalline hydroxyapatite, On the other hand, the time course of conversion should be fast enough to have surgical utility. One approach is to combine a precursor ACP containing an inhibitor of crystal formation (e.g., the ACP of Example 1) with an ACP that does not contain an inhibitor of crystal formation (e.g., a promoter). The reactants may be mixed in a dry state, with the appropriate particulate size and an excess of the inhibitor-containing ACP. The reactants can then be exposed to crystal-forming conditions such as the addition of water, followed by an elevation in temperature (e.g., as occurs following introduction into the body), to convert the reactants to the PCA material of the invention. Other methods of controlled conversion involve the use of catalysts.

ACP precursor plus additional calcium phosphate sources: ACP may be reacted with a second calcium source (including a second ACP) using any reaction-promoting technique. In preferred embodiments; the second calcium source is itself a promoter. The reaction being promoted is the conversion of an amorphous calcium phosphate into a hardened nanocrystalline or poorly crystalline apatitic calcium phosphate. Such reactions include acid/base, displacement, substitution, and hydrolysis reactions as well as purely physical and mechanical reactions (e.g., grinding, mixing). Catalytic conversion, such as surface-catalyzed conversion of ACP to a PCA material, may also be employed. Under any reaction scheme, it is important that the ACP retains significant amorphous character throughout the reaction. Specifically, the overall crystallinity within the starting ACP should not exceed that desired in the end product. Thus, certain reaction schemes may require stabilization of the amorphous nature of the ACP throughout the reaction period. Examples of inhibitors of crystal formation that are known to the art and are useful for such stabilization include carbonate, pyrophosphate and magnesium.

In some preferred embodiments, the ACP component is activated under heat in order to facilitate the conversion being promoted by the second calcium containing reactant or other promoter. Examples of suitable such second reactant promoters include DCPD, other crystalline or poorly crystalline calcium phosphates, calcium sources, phosphate sources, or a second ACP. Other methods of promoting conversion, such as catalysis of the use of ionic solvents or promoters of nucleation, may also be employed to promote reaction between substituents. The second calcium phosphate reactant may be of any crystalline structure and should be chosen so as to be reactive with the first ACP either directly or through the use of reaction enhancing vehicles such as ionic solvents or catalysts. Appropriate reaction conditions will be determined by demonstration of rapid hardening at 37° C. after the reactants are mixed and water is added.

The delivery vehicle formation reaction may also be designed to produce an end product that is porous. In one embodiment, the use of a dry mixture of controlled particle size reactants leads to a porous material. Other methods of promoting porosity, such as chemical or physical etching and leaching, may be employed.

The present invention provides a novel process for activating a standard amorphous calcium phosphate precipitate into highly reactive amorphous solids. The amorphous solids can be used in the reaction described above to form a poorly- or nanocrystalline synthetic apatitic calcium phosphate that provides bioactivity, bioresorbabiity and structural integrity. The novel amorphous material can be reacted with other calcium phosphates at or below 37° C. to form a bone-like material consisting of poorly crystalline apatitic calcium phosphate.

Prior art acid-base reactions of conventional crystalline calcium phosphates produce poorly reacted solids, having reaction products that are too crystalline to be sufficiently resorbable in living tissues. The reactions from the prior art generally incomplete and the reaction products are inhomogeneous. In constrast, the amorphous calcium phosphate of the present invention reacts quickly and completely with a wide variety of calcium phosphates and other calcium- or phosphorus-bearing materials to provide a homogeneous product.

The source of the enhanced reactivity of the ACP of the present invention is not completely understood; however, it is believed to be associated with the amorphicity (lack of crystallinity) and, in some embodiments, ion pair site vacancies in the material, as created by the process of the present invention. The vacancies may provide reactive sites for subsequent reaction. These observations will be discussed more fully, below.

The method of the present invention permits initial formation of amorphous calcium phosphate particles of less than 1000 Å, preferably 200-500 Å, and most preferably 300 Å, the further growth of which is curtailed by rapid precipitation of the product from solution. During reaction of calcium and phosphate ion sources to form an amorphous calcium phosphate, a third ion is introduced in the solution so that this third ion is incorporated in the amorphous precipitate structure instead of trivalent PO₄^3-group(s). Because some PO₄^3-is replaced by the third ion, the overall PO₄^3-decreases, thus increasing the Ca/P ratio of the amorphous precipitate (as compared to standard amorphous calcium phosphate) and modifying the valence or charge sate of the calcium phosphate. The amorphous solids then may be rapidly freeze-dried to preserve the chemical and physical properties of the material. The amorphous solids then may be treated under specific conditions selected to promote removal of at least some of the third ion. Where the third ion is carbonate, specific temperature and pressure conditions lead to the reduction of total carbon, presumably as gaseous carbon dioxide from the amorphous solid, while maintaining the product amorphicity.

The resultant material is an amorphous solid with a higher Ca/P ratio than is typically found in amorphous calcium phosphates, where the ratio generally reported in the past is 1.50. Further, removing carbon from the material results in a vacancies in the interstitial structure within the amorphous solids, rendering it a highly reactive solid. There may be several possible vacancies sources. The material possess a porosity which promotes reactivity by various means, such as increased surface area. The material may also undergo a change in the stoichmetry balance upon removal of the third ion. This stoichiometry change may result a charge imbalance which is responsible for the increased reactivity of the amorphous calcium phosphate.

It is desirable to maintain substantial amorphous character within the material throughout the entire process. If crystallinity in its entirety (single crystalline regions), or even in local domains (microcrystalline regions), is introduced to excess during the process or in the final product, the solid has been found to be less reactive. The resultant highly reactive calcium phosphate is amorphous in nature and has a calcium to phosphorous ratio in the range of 1.55 to 1.65. In a preferred embodiment, the amorphous calcium phosphate has a Ca/P ratio of about 1.58.

The amorphous state of the amorphous calcium phosphate is induced by controlling the rate and duration of the precipitation process. The amorphous calcium phosphate of the present invention is precipitated from solution under conditions where initial precipitation is rapid. Rapid precipitation results in the formation of many extremely small calcium phosphate nuclei. Additionally, rapid crystal or grain growth leads to the production of more defects within each grain, thereby also increasing solubility. At the extreme end of the spectrum, crystal or grain growth is so rapid and defect density is so significant that an amorphous calcium phosphate results. Amorphous calcium phosphate is gel-like and includes solid solutions with variable compositions. These gels have no long range structure, but are homogeneous when measured on an Angstrom scale. Under physiological conditions, these amorphous compounds have high solubilities, high formation rates and high rates of conversion to poorly crystalline apatitic calcium phosphate.

The amorphous calcium phosphate solids acquired by this method retain their amorphous nature sufficiently long enough to be introduced into the final reaction as substantially amorphous solids. They can also be mixed and reacted with other solids or solutions containing phosphates, to obtain solids containing a homogeneous distribution of nanometer-sized crystals. Further, in preferred embodiments, because the amorphous calcium phosphate reacts completely with the other solids, the Ca/P of the resultant solid will constitute the total calcium and phosphorous from such reaction, i.e., there will be an essentially complete reaction. When a proper molar concentration of phosphate from the solution or solids is reacted with the novel amorphous calcium phosphate material, a poorly crystalline apatitic calcium phosphate material (Ca/P 1.1-1.9) is obtained. Thus, the present invention permits one to design and modify the chemical composition of the resultant product, thereby providing a further mode of controlling bioactivity of the final product used as a delivery vehicle or cell scaffold.

In one embodiment of the present invention, a solution is prepared that contains calcium and phosphate ions and a third ion in a concentration, at a pH, and at a temperature that will promote the rapid nucleation and precipitation of calcium phosphate. When precipitation is sufficiently rapid, an amorphous gel-like calcium phosphate is formed. Because the thermodynamically favored crystalline form of hydroxyapatite is enhanced by reducing the rate of reaction, certain processing steps of increasing the rate of reaction may be taken to ensure than an amorphous compound is obtained. The following factors, among others, are to be considered when designing a solution for the rapid precipitation of the amorphous calcium phosphate of the present invention.

Preferred conditions: Rapid mixture of calcium and phosphate sources to increase the rate of reaction. The rate of reaction is increased to favor non-stable phases as a product. Allowing more reaction time for each of the ions to juxtapose correctly to form a solid will result in a more thermodynamically favorable crystalline and stable structure.

Preferred calcium and phosphate sources: The use of highly concentrated or near supersaturation solutions ensures that a more rapid reaction will occur.

Preferred temperature: Although the reaction can be carried out at room temperature, temperatures of near boiling point to increase the concentration of one reactant is a possible means of increasing the rate of reaction.

In one embodiment calcium ions, phosphate ions and carbonate ions are mixed together rapidly in an aqueous solution to obtain a carbonate containing amorphous calcium phosphate solid. The relative concentrations of the ions are selected to give a precipitate having the desired Ca/P ratio. The carbonate ion substitutes for a phosphate ion in the amorphous calcium phosphate. The carbonated amorphous calcium phosphate may be obtained by precipitation from an aqueous carbonate solution. Suitable aqueous carbonate solutions include, by way of example only, bicarbonate solution, sodium carbonate solution, potassium carbonate solution. It is further contemplated as within the scope of the invention to use non-aqueous solutions.

Use of a carbonated material is desirable because it permits manipulations of the Ca/P ratio by substitution of PO₄^3- by CO₃^2-. Additionally, the presence of CO₃^2- is known to retard the development of crystallinity in amorphous calcium phosphate. Is recognized, however, that other ions or a mixture of ions may be suitable in place of or in addition to carbonate ion in modifying the Ca/P ratio and in introduction of reactive site vacancies into the amorphous calcium phosphate, such as by way of example only, nitrate, nitrite, acetate, Mg⁺²and P₂O₇^4- ions.

The amorphous calcium phosphate precipitate may be collected and filtered prior to activation. It is preferred to perform this step in a cold room or at sub-ambient temperatures so as to preserve the amorphous state of the precipitate collected. Collection may typically be carried out by any conventional means, including, but in no way limited to, gravity filtration, vacuum filtration or centrifugation. The collected precipitate is gelatinous and is washed more than once with distilled water.

The washed precipitate is then dried under any condition that maintain the amorphous character of the material. Lyophilization is a suitable, but not exclusive, technique. The precipitate is frozen and, while being kept frozen, is dried to remove the bulk of the entrained liquid. This procedure may be accomplished by placing the frozen precipitate into a vacuum chamber for a given period of time. Freeze-drying typically occurs at liquid nitrogen temperatures for a time in the range of 12-78 hrs, preferably about 24 hours, and under a vacuum in the range of 10^--10^-4, preferably 10^-4, torr. A preferred method includes lyophilizations because the cryogenic temperatures typically used in lyophilization inhibit further crystallization of the material. As a result, the amorphous calcium phosphate obtained thereby is an extremely fine free flowing powder.

The dried ACP may then be activated. In a preferred embodiment, where carbonate is present in the ACP, the ACP powder is heated to drive off remaining free water and water of hydration and to remove carbon, presumably through the decomposition of CO₃^2- into CO₂ and oxygen. The heating step is carried out at a temperature of less than 500-600° C. but more than 425° C., so as to prevent conversion of the amorphous calcium phosphate into crystalline hydroxypatite. Heating is preferably carried out at a temperature in the range of 450-460° C., preferably for ½ hour to 6 hours.

Low crystallinity and site vacancies (porosity and/or stoichiometric changes) may account for the observed higher reactivity of the activated amorphous calcium phosphate of the present invention. This is exemplified by the following observations. A carbonate-containing amorphous calcium phosphate which has been heated to 525° C. is observed to have an increase in formation of crystalline hydroxyapatite and to have a corresponding decrease in reactivity. Amorphous calcium phosphate that is heated to only 400° C. retains its amorphous characteristic, but exhibits a decreased reactivity. Presumably this decrease in reactivity is related to the higher carbonate levels (and fewer site vacancies) observed by IR in samples treated at this lower temperature. These findings suggest that both amorphicity and decreased carbon content (vacant reactive sites) are a factor in reactivity. This is not limited to be in any way an exclusive basis for reactivity. Other bases for the observed reactivity are considered to be within the scope of the invention. The resulting amorphous calcium phosphate powder is a highly reactive amorphous calcium phosphate material with a Ca/P ratio of between 1.1-1.9, preferably about 1.55 to 1.65, and most preferably about 1.58. The powder has been characterized by a variety of analytical techniques.

In FIG. 1, a high-resolution transmission electron micrograph is shown to illustrate the morphological characteristics and the angstrom-sized nature of the preferred reactive amorphous calcium phosphate of the present invention. Preferred particle sizes are less than 1,000 Å. Note the unclear boundaries separating the globule-like clusters, lacking clear edges and surfaces, in contrast to crystalline materials.

The amorphous nature of the reactive ACP of the invention is characterized by an X-ray pattern that is devoid of sharp peaks at any position of the diffracting angles that correspond to known crystalline calcium phosphates (FIG. 4a). The Ca/P measurement performed using wave length-dispersive X-ray analysis on an electron micro-probe of the same material after heat treatment yields Ca/P to be 1.58 (FIG. 2).

These characterizations demonstrate that although there is a change in the local moiety of certain groups in the amorphous calcium phosphate solids, the overall amorphicity is maintained throughout the process.

In another preferred embodiment, the highly reactive amorphous calcium phosphate is reacted with a second calcium phosphate to obtain a PCA material. As discussed above, crystalline hydroxyapatite is the thermodynamically preferred reaction product, and is usually described as not resorbable under physiological conditions. The use of an amorphous calcium phosphate, which can convert quickly and completely to produce an apatitic compound without significant crystallization, provides a novel route to a poorly-crystalline apatitic calcium phosphate that is resorbable under physiological conditions.

The amorphous calcium phosphate powder of the present invention may be mixed with a promoter and thereby convert to form a PCA material. This reaction may occur at room temperature upon mixing of the powder with any of a variety of both acidic and basic calcium phosphates in the presence of a limited amount of a fluid such as, but not limited to, water, saline, buffer solution, serum or tissue culture medium. Depending upon the amount of fluid added, the mixture of amorphous calcium phosphate of the present invention and a second calcium phosphate results in a highly formable and/or highly injectable paste with varying degrees of paste consistency.

The method of preparation of the promoter and/or the ACP will affect the ease by which the hydrated precursor is converted into the PCA material. As noted above, the method of mixing the powdered reactants prior to addition of liquid affects the reactivity of the system. Thus, hand mixing using a mortar and pestle does not result in as reactive a system as a prolonged machine grinding of the reactant powders. Therefore when comparing promoters, it is important to use standardized preparation conditions.

It is hypothesized that the conversion of ACP to the reactive PCA calcium phosphate is a surface catalyzed phenomenon. If so, it may be desirable to produce a particular promoter with a reproducible surface area. Specific surface area of the ACP and promoter powders can be controlled to control the reaction conditions and final PCA material properties. Thus, to control reaction reproducibility it is advantageous to provide a promoter with a known grain size distribution. Standard sieving techniques are suitable for selection of specific grain sizes. Surface area has been shown to be correlated to the compressive strength, and possibly the porosity and resorbability, of the PCA material.

Man calcium- or phosphate-containing compounds may be used as participating promoters in the hardening reaction. A calcium phosphate promoter, may be of any crystalline structure and should be chosen so as to be reactive with ACP either directly or through the use of enhancing promoters. Preferred participating promoters are those which tend themselves to undergo conversion to hydroxyapatite through an intermediate PCA calcium phosphate phase.

Appropriate calcium phosphates for use as promoters with the ACP described herein include neutral, basic, and acidic calcium phosphates, preferably apatitic phosphates, that provide the appropriate stoichiometry for reaction to obtain a apatitic calcium phosphate. In a preferred embodiment, an acidic (pH 5-7) calcium phosphate is used. Suitable calcium phosphates include, but are in no way limited to, calcium metaphosphate, dicalcium phosphate dihydrate, heptacalcium decaphosphate, tricalcium phosphates, calcium pyrophosphate dihydrate, the poorly crystalline apatitic material of the invention, calcium pyrophosphate, octacalcium phosphate, tetracalcium phosphate and additional ACPs. Other solids that would provide a source of phosphate or calcium, such as, by way of example only, CaO, CaCO₃, calcium acetate, and H₃PO₄, may be mixed to form a final product to yield a desired Ca/P ratio close to about 1.1-1.9, preferably about 1.3 to 1.5. It may be desirable to provide the second component in the amorphous or poorly crystalline state, as well.

Some calcium phosphate promoters may be prepared as either weak promoters or strong promoters. For instance, a DCPD sample with a grain size in the range of 100-125 μm (or distribution B3 in Example 5) reacts only marginally with the highly reactive ACP of the invention under certain conditions (see Example 5). DCPD of this grain size may be considered "weakly promoting". Thus, DCPD may be used in this format to screen for highly reactive ACPs.

In some embodiments of the invention, it is not required that the reaction employ a participating second calcium phosphate to produce a PCA material. Rather, it is within the scope of the invention to merely promote hardening and the conversion of the reactive ACP into a PCA material by addition of one or more "passive" promoters (also termed "non-reactive" or "non-participatory" promoters) that do not participate in the reaction. Suitable passive promoters include, but are not limited to, materials or treatments that have previously been described as promoting conversion of calcium phosphate materials into hydroxyapatite. For example, water, heat, nucleators and catalysts can be used as passive promoters. In some embodiments, the catalysts provide surface area, the presence of which promoters the hardening and conversion of ACP to poorly crystalline apatitic calcium phosphate. For example, Al₂O₃, mica, glass and sand, among other things, are useful passive promoters. In preferred embodiments, material promoters are employed that are insoluble or of low solubility in water, may be prepared in granular form in the range of 1-200 μm in diameter and are resorbable in vivo. Thus, polymers such as poly L-lactic acid (PLLA) and polyglycolic acid (PGA) are particularly desirable promoters.

Where a second calcium phosphate is employed as a promoter, it is often crystalline, as is evidenced by the presence of sharp diffraction peaks typical to the calcium phosphate of interest in the X-ray diffraction pattern (FIG. 4b). In contrast, the reactive ACP is amorphous and shows no identifiable peaks by X-ray diffraction (FIG. 4a). Despite its higher crystallinity, however, X-ray diffraction suggests that dicalcium diphosphate is consumed in the reaction with reactive ACP and the product PCA material is of much reduced crystallinity. Similarly, when stoichiometric HA is employed as a second calcium phosphate source, it is also consumed in the reaction and a PCA material of reduced crystallinity is produced.

Because at least one of the reactants is amorphous and highly reactive, the formation reaction of the present invention proceeds at or above room temperature to provide a hardened apatitic material having a poorly-crystalline or microcrystalline microstructure. In preferred embodiments, the conversion reaction also is substantially complete, thereby insuring that all calcium and phosphate of the mixture are consumed by the resultant PCA product. This result permits reliable manufacture of apatitic products simply by selection of the relative proportions of the starting amorphous and secondary calcium phosphates. It is desirable to maintain a calcium to phosphate ratio of about 1.2-1.68, preferably less than 1.5, and most preferably about 1.38.

The product apatitic material contains labile environments characteristic of naturally-occurring bone. In naturally occurring bone, minerals are characterized by nanometer-sized structure, providing high surface areas to interact with the surrounding tissue environment, resulting in resorption and remodelling of tissues. The present invention, with its nanometer-sized crystals as the product, closely mimics the naturally occurring bone materials. Further, properties such as crystallinity and Ca/P ratios are closely designed in the present invention to simulate the mineral properties found in living tissues of bone.

The PCA produced during the inventive reaction is associated with hardening of the hydrated precursor material. It should be noted, however, that while complete conversion of the ACP precursor is a preferred embodiment, hardening of the hydrated precursors may occur prior to complete conversion or even in the absence of complete conversion. Such partially converting, but nonetheless hardening, reactions are considered to be within the scope of the invention.

As mentioned above, combination of dry ACP with any other reactants and a limited amount of aqueous solution produces a hydrated precursor. By selecting the appropriate amount of liquid to be added to the reactants, the viscosity of the may be adjusted according to need. The hydrated precursor may be prepared either with an injectable or a formable consistency. Injectable consistency means as thick as possible while still capable of passing through a 16 to 18 gauge needle. Most often, this will be a "toothpaste"-like consistency. Formable refers to consistency that allows the material to retain its shape. In the extreme case of a formable consistency, the hydrated precursor will have the consistency of glazing putty or caulking compounds. The hydrated precursor also may be prepared with just enough liquid to be both injectable and formable. In the past form, the material has markedly improved flow characteristics over prior art compositions. Flow characteristics are toothpaste-like while prior art materials generally exhibit a granular or oatmeal-like consistency. The hydrated precursor may be prepared before use, up to a period of several hours if held at room temperature and if evaporation is minimized. The storage time may be extended by maintaining the paste at reduced temperatures in the range of 1-10° C. in the refrigerator provided steps are taken to minimize evaporative loss.

In some preferred embodiments (e.g., Examples 9-14, below), the reaction is endothermic and occurs slowly at room temperature, but is accelerated significantly at body temperature. This is particularly useful in a surgical situation, since the paste formed by mixing reactants with water remains injectable for a considerable period of time (up to several hours) while held at or below room temperature. Thus, at room temperature (ca. 22° C.) the paste hardens after a timer greater than one hour and remains formable and/or injectable for longer than 10 minutes, preferably longer than one hour, and most preferably longer than three hours. Following injection at the implant site (ca. 37° C.), the past hardens in less than about an hour, preferably in about 10-30 minutes.

Composites and Additives

The PCA material of the instant invention may be formed as a composite with other substances. Composites may be desirable to change any number of physical parameters of the vehicle including but not limited to strength, resorption time, adherence, injectability, frictional characteristics, or therapeutic agent carrying capacity or release kinetics. In general, those practiced in the art of composite fabrication will understand the methods and concepts important in composite fabrication. Additional guidance for the preparation of PCA material composites may be obtained in co-pending United States patent application entitled "Bioresorbable Ceramic Composites", filed on even date herewith and incorporated herein by reference.

In vitro Implant Formation

In addition to surgical application in paste form, the inventive implants may be pre-formed outside the body, hardened, and implanted in the solid form. Pre-formed devices may be hand shaped, molded or machined. Loading of the therapeutic agent may be accomplished by addition of the agent directly to the buffer or vehicle used to prepare the hydrated precursor. Alternatively, after hardening, the vehicle may be exposed to the therapeutic agent using dipping, rolling or spray coat methods.

Biologically Active Agents

Any biologically useful agent may be delivered from the inventive PCA material implant. In general, the only requirement is that the substance remain active in the presence of the material during fabrication or be capable of being subsequently activated or re-activated. Since the inventive paste can be prepared with a large number of aqueous vehicles and substituents, those in the art will be familiar with which specific additives can be included in order to improve stability of the agent. The stability and/or compatibility of a particular agent with the inventive material, as well as fabrication strategies, may be tested empirically in vitro. Specifically, the agent may be incorporated into the inventive material by one or more of the of the methods described herein. Following hardening of the vehicle at 37° C., the substance may be leached from the material into an analysis medium such as water or an appropriate buffer and the compound collected from the Material by diffusion into the analysis medium. The analysis medium may then be analyzed for the presence of active agent. In some instances, the material will be broken up, pulverized, or otherwise fragmented prior to contacting the analysis medium. Other methods of analysis that do not require agent diffusion, such as the growth of cells on the material or other physical, chemical, or biological assays will be known to practitioners for specific compounds.

Biologically active agents useful in the practice of the present invention include any substance having biologically activity, including organic molecules, proteins, peptides, nucleic acids, nucleoproteins, polysaccharides, glycoproteins, lipoproteins, and synthetic and biologically engineered analogs thereof. Also included are chemical agents that have biological effects (e.g., antibiotics, dyes, etc.). Proteins can be prepared by synthetic, biochemical, or recombinant techniques. Preferably, though not necessarily, the biologically active agent is one that has been deemed safe and effective for use by an appropriate governmental agency or body. For example, drugs approved for human use in the United States are listed by the Food and Drug Administration (FDA) under 21 C.F.R. §§330.5, 331-361, and 440-460; drugs approved for veterinary use in the United States are listed by the FDA under 21 C.F.R. 517 §500-582.

The term "biologically active agent" includes pharmacologically active substances that produce a local or systemic effect in animals, plants, or viruses. The term thus means any substance intended for use in the diagnosis, cure, mitigation, treatment or prevention of disease or in the enhancement of desirable physical or mental development and conditions in an animal, plant, or virus. The term "animal" used herein is taken to mean mammals, such as primates (including humans), sheep, horses, cattle, pigs, dogs, cats, rats, and mice; birds; fish; insects; arachnids; protists (e.g. protozoa); and prokaryotic bacteria.

Classes of biologically active compounds that can be loaded into the delivery vehicle of the present invention include, but are not limited to, anti-AIDS substances, anti-cancer substances, antibiotics, ACE inhibitors, antigens, adrenegic antagonists, antacids, immunosuppressants, anti-viral substances, enzyme inhibitors, neurotoxins, opioids, hypnotics, antihistamines, lubricants, tranquilizers, anti-convulsants, muscle relaxants and anti-Parkinson substances, anti-spasmodics and muscle contractants, anti-diarrheals, anti-emetics, laxatives, diuretics, miotics and anti-chloinergics, anti-glaucoma compounds, anti-parasite and/or anti-protozoal compounds, anti-hypertensives, analgesics, anti-pyretics, anti-inflammatory agents, anti-histamines, anti-tussive agents, anti-vertigo, antinertigic and anti-motion sickness medications, local anesthetics, ophthalmics, prostaglandins, anti-depressants, anti-psychotic substances, anti-emetics, imaging agents, specific targeting agents, trophic factors, growth factors, immunosuppressants, immunoactivators, anti-mitotics neurotransmitters, proteins, cell response modifiers, vaccines, nucleic acids, genes, gene fragments, gene regulatory sequences (such as promoters, enhancers, or other regulatory sites), antisense molecules, and other bioactive moieties or components of biosynthetic pathways.

A more complete listing of classes of compounds suitable for loading into delivery vehicles according to the present invention may be found in the Pharmazeutische Wirkstoffe (Von Kleeman et al. (eds), Stuttgart, N.Y. 1987), or in any of a variety of available pharmacology textbooks, such as Lippincott's Illustrated Pharmacology Reviews (Harvey et al. (eds), J. B. Lippincott Co., Philadelphia, 1992) or Examination & Board Review Pharmacology (Kratzing et al., Appleton & Lange, Connecticut, 1993), each of which is incorporated herein by reference. Examples of particular biologically active substances are presented below:

Angiogenic factors are substances that stimulate the growth of vasculature and include compounds such as veg-f, and some growth factors and mitogens.

Anti-AIDS substances are substances used to treat or prevent Autoimmune Deficiency Syndrome (AIDS). Examples of such substances include CD4, 3′-azido-3′-deoxythymidine (AZT), 9-(2-hydroxyethoxymethyl)-guanine acyclovir(), phosphonoformic acid, 1-adamantanamine, peptide T, and 2′,3′-dideoxycytidine.

Anti-cancer substances are substances used to treat or prevent cancer. Examples of such substances include anti-metabolites (such as, for example, methotrexate, fluorouracil, 5-fluorouracil, cytarabine, mercaptopurine, 6-mercaptopurine, 6-thioguanine), antibiotics (such as, for example, daunorubicin, doxoubicin), alkylating agents (such as for example, mechlorethamine, cyclophosphamide, uracil mustard, busulfan, carmustine, lomusline), mitotic spindle poisons (such as, for example, vinblastine, vincritine), hormones (such as, for example, hydroxyprogesterone, medroxyprogesterone acetate, magistral acetate, diethylstilbestrol, testosterone propionate, fluoxymesterone), and other agents (such as, for example, vindesine, hydroxyurea, procarbazine, aminoglutethimide, melphalan, chlorambucil, acarbazine (DTIC: dimethyltriazenomidazole carboxamide), cytosine arabinoxide).

Antibiotics are art recognized and are substances which inhibit the growth of or kill microorganisms. Antibiotics can be produced synthetically or by microorganisms. Examples of antibiotics include bactericidal agents, such as aminoglycosides (e.g., gentamicin, tobramycin, netilmicin, streptomycin, amikacin, neomycin), bacitracin, corbapenems (e.g., imipenem/cislastatin), cephalosporins, colistin, methenamine, monobactams (e.g., aztrenam), penicillins (e.g., penicillin G, penicillin V, methicillin, natcillin, oxacillin, cloxacillin, dicloxacillin, ampicillin, amoxicillin, carbenicillin, ticarcillin, piperacillin, mezlocillin, azlocillin), polymyxin B, quinolones, and vancmycin; and bacteriostatic agents such as chloramphenicol, clindamyan, macrolides (e.g., erythromycin, azithromycin, clarithromycin), lincomyan, nitrofurantoin, sulfonamides, tetracyclines (e.g., tetracycline, doxycycline, minocycline, demeclocyline), and trimethoprim. Also included are metronidazole, fluoroquinolones, and ritampin. Antibiotics are sometimes provided in insoluble form, which can be used where delayed delivery is desired.

Anti-viral agents are substances capable of destroying or suppressing the replication of viruses. Examples of anti-viral include a-methyl-P-adamantane methylamine, 1,-D-ribofuranosyl-1,2,4-triazole-3 carboxamide, 9-[2-hydroxy-ethoxy]methylguanine, adamantanamine, 5-iodo-2′-deoxyuridine, trifluorothymidine, interferon, and adenine arabinoside. Particular agents useful in the treatment of herpes viruses include acyclovir, vidarabine, idoxuridine, and ganciclovir.

Enzyme inhibitors are substances which inhibit an enzymatic reaction. Examples of enzyme inhibitors include edrophonium chloride, N-methylphysostigmine, neostigmine bromide, physostigmine sulfate, tacrine HCl, tacrine,1-hydroxy maleate, iodotubercidin, p-bromotetramisole, 10-(alpha-diethylaminopropionyl)-phenothiazine hydrochloride, calmidazolium chloride, hemicholinium-3,3,5-dinitrocatechol, diacylglycerol kinase inhibitor I, diacylglycerol kinase inhibitor II, 3-phenylproparglyamine, N⁶-monomethyl-L-arginine acetate, carbidopa, 3-hydroxybenzylhydrazine HCl, hydralazine HCl, clorgyline HCl, deprenyl HCl,L(-)-, deprenyl HCL,D(+)-, hydroxylamine HCl, iproniazid phosphate, 6-MeO-tetrahydro-9H-pyrido-indole, nialamide, pargyline HCl, quinacrine HCl, semicarbazide HCl, tranylcypromine HCl, N,N-diethylaminoethyl-2,2-diphenylvalerate hydrochloride, 3-isobutyl-1-methylxanthane, papaverine HCl, indomethacind, 2-cyclooctyl-2-hydroxyethylamine hydrochloride, 2,3-dichloro-a-methylbenzylamine (DCMB), 8,9-dichloro-2,3,4,5-tetrahydro-1H-2-benzazepine hydrochloride, p-aminoglutethimide, p-aminoglutethimide tartrate,R(+)-, p-aminoglutethimide tartrate,S(-)-, 3-iodotyrosine, alpha-methyltyrosine,L-, alpha-methyltyrosine,D L-, acetazolamide, dichlorphenamide, 6-hydroxy-2-benzothiazolesulfonamide, and allopurinol.

Neurotoxins are substances which have a toxic effect through their action on the nervous system, e.g. nerve cells. Neurotoxins include adrenergic neurotoxins, chloinergic neurotoxins, dopaminergic neurotoxins, calcium channel blockers, and other neurotoxins. Examples of adrenergic neurorotoxins include N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride. Examples of cholinergic neurotoxins include acetylethylcholine mustard hydrochloride. Examples of dopaminergic neurotoxins include 6-hydroxydopamine HBr, 1-methyl-4-(2-methylphenyl)-1,2,3,6-tetrahydro-pyridine hydrochloride, 1-methyl-4-phenyl-2,3-dihydropyridinium perchlorate, N-methyl-4-phenyl-1,2,5,6-tetrahydropyridine HCl, 1-methyl-4-phenylpyridinium iodide. Examples of calcium channel blockers include Ω-conatoxin and verapamil.

Opioids are substances having opiate like effects that are not derived from opium. Opioids include opioid agonists and opioid antagonists. Opioid agonists include codeine sulfate, fentanyl citrate, hydrocodone bitartrate, loperamide HCl, morphone sulfate, noscapine, norcodeine, normophine, thebaine. Opioid antagonists include nor-binaltorphimine HCl, buprenorphine, chlornaltrexamine 2HCl, funaltrexamione HCl, nalbuphine HCl, nalorphine HCl, naloxone HCl, naloxonazine, naltrexone HCl, and naltrindole HCl.

Hypnotics are substances which produce a hypnotic effect. Hypnotics include pentobarbital sodium, phenobarbital, secobarbital, thiopental and mixtures, thereof, heterocyclic hypnotics, dioxopiperidines, glutarmides, diethyl isovaleramide, a-bromoisovaleryl urea, urethanes and disulfanes.

Antihistamines are substances which competitively inhibit the effects of histamines. Examples include pyrilamine, chloropheniramine, tetrahydrazoline, and the like.

Lubricants are substances that increase the lubricity of the environment into which they are delivered. Examples of biologically active lubricants include water and saline.

Tranquilizers are substances which provide a tranquilizing effect. Examples of tranquilizers include chloropromazine, promazine, fluphenzaine, reserpine, deserpidine, and meprobamate.

Anti-convulsants are substances which have an effect of preventing, reducing, or eliminating convulsions. Examples of such agents include primidone, phenytoin, valproate, Chk and ethosuximide.

Anti-inflammatories are compounds that inhibit inflammation. Different types of anti-inflammatory drugs block different chemical mediators. Examples of anti-inflammatory agents include nonsteroidal anti-inflammatory drugs (NSAIDS), such as aspirin, phenylibutazone, indomethacin, sulindac, tolmetin, ibuprofen, piroxicam, fenamates, which have anti-inflammatory, analgesic, and antipyretic activities. Also included are non-narcotic analgesics such as acetaminophen and phenacetin, although the anti-inflammatory activity of these drugs is weaker. Certain slow-acting anti-inflammatories, such as gold salts, chloroquine, D-Penicillamine, and methotrexate are useful in the treatment of arthritis. Gout-specific anti-inflammatories include colchicine, allopurinol, probenecid, and sulfinpyrazone.

Muscle relaxants and anti-Parkinson agents are agents which relax muscles or reduce or eliminate symptoms associated with Parkinson's disease. Examples of such agents include mephenesin, methocarbomal, cyclobenzaprine hydrochloride, trihexylphenidyl hydrochloride, levodopa/carbidopa, and biperiden.

Anti-spasmodics and muscle contractants are substances capable of preventing or relieving muscle spasms or contractions. Examples of such agents include atropine, scopolamine, oxyphenonium, and papaverine.

Miotics and anti-cholinergics are compounds which cause bronchodilation. Examples include echothiophate, pilocarpine, physostigmine salicylate, diisopropylfluorophosphate, epinephrine, neostigmine, carbachol, methacholine, bethanechol, and the like.

Anti-glaucoma compounds include betaxalol, pilocarpine, timilol, timilol salts, and combinations of timolol, and/or its salts, with pilocarpine.

Anti-parasitic, -protozoal and -fungals include ivermectin, pyrimethamine, trisulfapyrimidine, clindamycin, amphotericin B, nystatin, flucytosine, ketocanazol, fluconazole, natamycin, miconazole, metronidazole, diloxanide furoate, paramomycine, chlorquine, emetine, dehydroemetine, sodium stibogluconate, (for leishmaniasis), melarsoprol (for trypanosomiasis), nifurtimox (for trypanosomiasis), suramin (for trypanosomiasis), pentamidone (for trypanosomiasis), and anti-malarial agents (such as, for example, primaquine, chloroquine, quinine, mefloquine, pyrimethamine, and chloroquanide).

Anti-hypertensives are substances capable of counteracting high blood pressure. Examples of such substances include alpha-methyldopa and the pivaloyloxyethyl ester of alpha-methyldopa.

Analgesics are substances capable of preventing, reducing, or relieving pain and Anti-pyretics are substances capable of relieving or reducing fever. Examples of such substances include aspirin, phenybutazone, idomethacin, sulindac, tolmetic, ibuprofen, piroxicam, fenamates, acetaminophen, phenacetin, morphine sulfate, codeine sulfate, meperidine, and nalorphine.

Local anesthetics are substances which have an anesthetic effect in a localized region. Examples of such anesthetics include procaine, lidocain, tetracaine and dibucaine.

Ophthalmics include diagnostics agents such as sodium fluorescein, rose bengal, methacholine, adrenaline, cocaine, and atropine. Ophthalmic surgical additive include alpha-chymotrypsin and hyaluronidase.

Prostaglandins are art recognized and are a class of naturally occurring chemically related, long-chain hydroxy fatty acids that have a variety of biological effects.

Anti-depressants are substances capable of preventing or relieving depression. Examples of anti-depressants include imipramine, imitriptyline, nortriptyline, protriptyline, desipramine, amoxapine, doxepin, maprotiline, tranylcypromine, phenelzine, and isocarboxazide.

Anti-psychotic substances are substances which modify psychotic behavior. Examples of such agents include phenothiazines, butyrophenones and thioxanthenes.

Anti-emetics are substances which prevent or alleviate nausea or vomiting. An example of such a substance includes dramamine.

Imaging agents are agents capable of imaging a desired site, e.g. tumor, in vivo. Examples of imaging agents include substances having a label which is detectable in vivo, e.g. antibodies attached to fluorescent labels. The term antibody includes whole antibodies or fragments thereof.

Specific targeting agents include agents capable of delivering a therapeutic agent to a desired site, e.g. tumor, and providing a therapeutic effect. Examples of targeting agents include agents which can carry toxins or other agents which provide beneficial effects. The targeting agent can be an antibody linked to a toxin, e.g. ricin A or an antibody linked to a drug.

Neurotransmitters are substances which are released from a neuron on excitation and travel to either inhibit or excite a target cell. Examples of neurotransmitters include dopamine, serotonin, γ-aminobutyric acid, norepinephrine, histamine, acetylcholine, and epinephrine.

Trophic factors, growth factors, and cell response modifiers are factors whose continued presence improves the viability or longevity of a cell. In some cases, they produce chemotactic effects, or have protective effects against toxins or neurotoxins, or against neurodegeneration. Suitable such factors include, but are not limited to, platelet-derived growth factor (PDGF), neutrophil-activating protein, monocyte chemoattractant protein, macrophage-inflammatory protein, platelet factor, platelet basic protein, and melanoma growth stimulating activity; epidermal growth factor, transforming growth factor (alpha), fibroblast growth factor, platelet-derived endothelial cell growth factor, insulin-like growth factor, glial derived growth neurotrophic factor, ciliary neurotrophic factor, nerve growth factor, and bone growth/cartilage-inducing factor (alpha and beta), or other bone morphogenetic proteins.

Other cell response modifiers are the interluekins, interleukin inhibitors or interleukin receptors, including interleukin 1 through interleukin 10; interferons, including alpha, beta and gamma; hematopoietic factors, including erythropoietin, granulocyte colony stimulating factor, macrophage colony stimulating factor and granulocyte-macrophage colony stimulating factor; tumor necrosis factors, including alpha and beta; transforming growth factors (beta), including beta-1, beta-2, beta-3, inhibin, and activin; and bone morphogenetic proteins such as OP-1, BMP-2 and BMP-7.

Hormones include estrogens (such as, for example, estradiol, estrone, estriol, diethylstibestrol, quinestrol, chlorotrianisene, ethinyl estradiol, mestranol), anti-estrogens (such as, for example, clomiphene, tamoxifen), progestins (such as, for example, medroxyprogesterone, norethindrone, hydroxyprogesterone, norgestrel), antiprogestin (mifepristone), androgens (such as, for example, testosterone cypionate, fluoxymesterone, danazol, testolactone), and anti-androgens (such as, for example, cyproterone acetate, flutamide). Hormones are commonly employed for hormone replacement therapy and/or for purposes of birth control. Steroid hormones, such as prednisone, are also used as immunosuppressants and anti-inflammatories. Delivery of steroid hormones can be delayed by esterification. Thyroid hormones include triiodothyronne, thyroxine, propylthiouracil, methimazole, and iodixode. Pituitary hormones include corticotropin, sumutotropin, oxytocin, and vasopressin.

Nucleic acids are molecules, including DNA or RNA molecules, that comprise one or more nucleosides and/or nucleotides. Since calcium compounds are known to promote cell transfection and DNA uptake in some systems, it is anticipated that resorption of the present delivery device may improve transfection efficiency. Nucleic acid molecules can be delivered as vaccines or, for example, as antisense agents. Alternatively, DNA molecules can be prepared for use in gene therapy, in which molecules can correct or compensate for genetic errors in cells into which the DNA molecules are to be introduced.

Standard protocols and regimens for delivery of the above-listed agents are known in the art. Typically, these protocols are based or oral or intravenous delivery. To the extent that the present invention provides for alternate delivery modes, modification to these protocols may be appropriate.

Biologically active agents are introduced into a delivery vehicle served from PCA material of the present invention during or after its formation (see Examples 20-21). Agents may conveniently be mixed into the paste prior to setting. Alternatively, the vehicle may be shaped and hardened and then exposed to the therapeutic agent in solution. This particular approach is particularly well suited in proteins, which are known to have an affinity for apatitic materials. A buffer solution containing the biologically active agent may be employed, instead of water, as the aqueous solution in which the amorphous calcium phosphate is converted into the synthetic, poorly crystalline apatitic material of the present invention. Buffers may be used in any pH range, but most often will be used in the range of 5.0 to 8.0 in preferred embodiments the pH will be compatible with prolonged stability and efficacy of the desired therapeutic agent and, in most preferred embodiments, will be in the range of 5.5 to 7.4. Suitable buffers include, but are not limited to, carbonates, phosphates (e.g., phosphate buffered saline), and organic buffers such as Tris, HEPES, and MOPS. Most often, the buffer will be selected for it's biocompatibility with the host tissues and its compatibility with the therapeutic agent. For most applications of nucleic acids, peptides or antibiotics a simple phosphate buffered saline will suffice.

Biologically active agents are introduced into the vehicle in amounts that allow delivery of an appropriate dosage of the agent to the implant site. In most cases, dosages are determined using guidelines known to practitioners and applicable to the particular agent in question. It is generally preferred, for these agents that bind to a receptor, to achieve local levels approximately 1-2 fold higher than the dissociation constant of the receptor-agent complex. Loading levels, device size, and resorption properties can be determined empirically through the use of animal models and human efficacy studies, as is common in the pharmaceutical industry.

One of the advantages of the present delivery material, as compared with ceramic devices generally, and with calcium phosphate materials in particular, is that it can be formed under mild reaction conditions. For example, although calcium phosphate-based ceramics (e.g., hydroxyapatites) have been much studied as potential drug delivery materials because of their biocompatibility and known affinity for protein agents, such materials are typically prepared in processes that require have detrimental effects on many therapeutic agents. For example, some methods require sintering above 500° C., others require the use of acidic conditions, and still others require extended periods of time to grow crystals containing the therapeutic agent. By contrast, the present synthetic PCA drug delivery vehicle can be prepared at ambient temperatures and physiologically relevant pHs (see Example 4). Accordingly, a wide variety of biologically active materials that might be destroyed during the preparation of standard calcium phosphate materials can be incorporated into the drug delivery material of the present invention. Protein agents in particular are often sensitive to heat and other unfavorable conditions; the present synthetic PCA material therefore constitutes a particularly improved delivery system for protein agents.

Cells

Where the PCA material of the invention is to be utilized in a cell seeding application, the hydrated precursor is preferably prepared with an aqueous solution that is a physiological medium. Examples of such media are well known in the art (e.g., Dulbecco's minimal essential medium; phosphate buffered saline; and carbonate, TRIS, or HEPES-buffered solutions); and those of ordinary skill are aware of particular media that are compatible with desired cell types.

Of course, it is not essential that the hydrated precursor be prepared with a buffered aqueous solution rather than water. However, as it is desirable to maintain cell viability, a hydrated precursor or hardened PCA material that has been prepared using water (or other minimal aqueous solution) will preferably be exposed to growth medium prior to, or at least coincident with, its exposure to cells. Introduction of a material into an animal can constitute exposure of the material to growth medium (and to cells).

The PCA material of the present invention may be prepared with any of a variety of additives, and/or may be prepared as a composite. For examples of desirable PCA material composites, see U.S. application entitled "Bioactive Ceramic Composites" and filed on even date herewith; for examples of biologically active materials that can be incorporated into the PCA material before or after cell seeding, see U.S. application entitled "Delivery Vehicle" and filed on even date herewith. In some cases, it will be particularly desirable to add factors to the PCA material that can affect cell growth, differentiation, and/or localization. For example, laminin, fibronectin, collagen, matrigel and its components, and other growth factors and extracellular matrix components.

Cells

The PCA material of the present invention may be seeded with any of a variety of cells. A "cell", according to the present invention, is any preparation of living tissue, including primary tissue explants and preparations thereof, isolated cells, cells lines (including transformed cells), and host cells. Preferably, autologous cells are employed, but xenogeneic, allogeneic, or syngeneic cells are also useful. Where the cells are not autologous, it may be desirable to administer immunosuppressive agents in order to minimize rejection. In preferred embodiments, such agents may be included within the seeded composition to ensure effective local concentrations of the agents and to minimize systemic effects of their administration. The cells employed may be primary cells, explants, or cell lines, and may be dividing or non-dividing cells. Cells may be expanded ex-vivo prior to introduction into the inventive PCA material. Autologous cells are preferably expanded in this way if a sufficient number of viable cells cannot be harvested from the host.

Any preparation of living cells may be use to seed the PCA material of the present invention. For example, cultured cells or isolated individual cells may be used. Alternatively or additionally, pieces of tissue, including tissue that has some internal structure, may be used. The cells may be primary tissue explants and preparations thereof, cell lines (including transformed cells), or host cells. Where the cells are host cells and are introduced into the inventive PCA material in vivo (see below), preferred sources of cells include, but are not limited to, the inner layer of the periosteum or perichondrium, blood or other fluids containing the cells of choice, and damaged host tissue (particularly bone or cartilage) that includes such cells.

Any available methods may be employed to harvest, maintain, expand, and prepare cells for use in the present invention. Useful references that describe such procedures include, for example, Freshney, Culture of Animal Cells: a Manual of Basic Technique, Alan R. Liss Inc., New York, N.Y., incorporated herein by reference.

The PCA material of the invention is useful as a scaffold for production of hard or soft tissues. Tissue-producing or -degrading cells that may be incorporated into the material include, but are not limited to, chondrocytes, osteocytes, osteoblasts, osteoclasts, mesenchymal stem cells, other bone- or cartilage-producing cells or cell lines, fibroblasts, muscle cells, hepatocytes, parenchymal cells, cells of intestinal origin, nerve cells, and skin cells.

Methods of isolating and culturing such tissue-producing or -degrading cells, and/or their precursors, are known in the art (see, for example, Vacanti et al., U.S. Pat. No. 5,041,138; Elgendy et al., Biomater. 14:263, 1993; Laurencin et al., J. Biomed. Res. 27:963, 1993; Freed et al., J. Cell. Biochem. 51:257, 1993; Atala et al., J. Urol. 150:745, 1993; Ishaug et al., J. Biomed. Mater. Res. 28:1445, 1994; Chu et al., J. Biomed. Mater. Res. 29:1147, 1995; Thomson et al., J. Biomater. Sci. Polymer Edn. 7:23, 1995, each of which is incorporated by reference).

For example, mesenchymal stem cells, which can differentiate into a variety of mesenchymal or connective tissues (including, for example, adipose, osseous, cartilagenous, elastic, and fibrous connective tissues), can be isolated, purified, and replicated according to known techniques (see Caplan et al., U.S. Pat. No. 5,486,359; Caplan et al., U.S. Pat. No. 5,226,914; Dennis et al., Cell Transplantation 1:23, 1992, each of which is incorporated herein by reference). Such mesenchymal cells have been studied in association with tricalcium phosphate and hydroxyapatite carriers and have been found to be capable of successful differentiation from within such carriers (see Caplan et al., U.S. Pat. No. 5,197,985, incorporated herein by reference). Similar procedures are employed to direct mesenchymal cell differentiation within PCA material scaffolds of the present invention.

Of course, the present invention is not limited to the use of tissue-producing cells. Certain preferred embodiments of the invention utilize such cells, primarily because the inventive PCA material is so well suited to tissue-regeneration applications (particularly with those involving growth of bone and/or cartilage). Any cell may be seeded into the PCA material of the invention. In some cases, it will be desirable to include other cells in addition with tissue-producing cells.

The cells that are seeded into the inventive PCA material may be genetically engineered, for example to produce a protein or other factor that is useful in the particular application. In preferred embodiments, cells may be engineered to produce molecules that impart resistance to host immune attack and rejection. The Fas-L and CR-1 genes are examples of useful such genes.

Other Components

When the inventive PCA material is used in a cell seeding application, one or more additives may be introduced into the PCA material before or after seeding. In certain preferred embodiments of the invention, one or more biologically active agents is incorporated into the PCA material. For discussion of such biologically active agents and their use in conjunction with the inventive PCA material, see U.S. application entitled "Delivery Vehicle" and filed on even date herewith.

Preferred biologically active agents for use in the seeded PCA material compositions of the present invention include factors that influence cell growth, differentiation, migration, and/or localization. For example, bone matrix contains a variety of protein factors that influence cell behavior (see, for example, Hubbell, Bio/Technology 13:565, 1995; Caplan et al., U.S. Pat. No. 4,609,551; Caplan et al., U.S. Pat. No. 4,620,327).

Also, cell matrix components can play important roles in division, differentiation, migration, and localization (see, for example, Hubbell, Bio/Technology 13:565, 1995). It may therefore be desirable to localize such matrix components within the seeded PCA material of the present invention. However, many of the functions achieved by association between cells and cell matrix components (e.g., definition of cell shape, achievement of cell polarity and organization, etc.) may well be accomplished by cell attachment directly to the inventive PCA material.

Other biologically active agents that are preferred for use in certain embodiments of the invention include nutrients, angiogenic factors, compounds that enhance or allow ingrowth of the lymphatic network or nerve fibers, etc. Immunomodulatory factors, and particularly inhibitors of inflammation, may be included where it is desirable to inhibit a host response to the implanted composition. Drugs may also be included.

Generally, cells are introduced into the PCA material of the present invention in vitro, although in vivo seeding approaches are employed in some circumstances (see below). Cells may be mixed with the hydrated precursor paste or putty prior to hardening or, alternatively, may be introduced into the PCA material composition after it has hardened. In either case, it is important that adequate growth (or storage) medium be provided to ensure cell viability. If the composition is to be implanted for use in vivo after in vitro seeding, sufficient growth medium must be supplied to ensure viability throughout, and for a short time following, the implant proceeding. Once the composition has been implanted, the porous nature of the PCA material allows the cells' nutritional requirements to be met by the circulating fluids of the host.

We have found Dulbecco's minimal essential medium to be particularly useful in the practice of the present invention. Other solutions that may be employed include, but are not limited to, phosphate-buffered saline; carbonate-, HEPES-, or TRIS-buffered solutions. In some cases, additional growth-stimulating components, such as serum, growth factors, amino acid nutrients, sugars, and salts, may be added to the aqueous solution employed in the present invention. However, it is generally desirable to avoid additives, as they can alter the hardening process of the inventive PCA material. If a particular collection of additives were selected to be used but had negative effects on PCA material characteristics, the precise PCA formulation can be varied and tested for its ability to satisfy hardening parameters in the presence of the additives.

Any available method may be employed to introduce the cells into the PCA material. In many cases, it will be desirable to introduce the cells into the hydrated precursor, before hardening. For example, cells may be injected into the hydrated precursor (preferably in combination with growth medium), or maybe introduced by other means such as pressure, vacuum, or osmosis. Alternatively (or additionally), cells may be layered on the hydrated precursor, or the hydrated precursor may be dipped into a cell suspension and allowed to remain there under conditions and for a time sufficient for cells to impregnate or attach to the material. Generally, it is desirable to avoid excessive manual manipulation of the cells in order to minimize cell death during the impregnation procedure. For example, in most situations it will not be desirable to manually mix or knead the cells with the PCA material paste; however, such an approach is perfectly useful in those cases in which a sufficient number of cells will survive the procedure. Cells may also be introduced into the hydrated precursor in vivo simply by placing the material in the body adjacent a source of desired cells. In some cases, it may be desirable to enhance such in vivo cell impregnation by including within the material an appropriate chemotactic factor, associative factor (i.e., a factor to which cells bind), or factor that induces differentiation of cells into the desired cell type.

Rather than being introduced into the hydrated precursor, cells may be introduced into the PCA material of the invention after it has hardened. Because the material is porous, cells are able to readily migrate into it. Cells may be introduced into the hardened PCA material by any available means. For example, cells may be layered on the material, or may be introduced by pressure, vacuum, or osmosis. Alternatively (or additionally), the hardened material may be placed in a cell suspension and maintained there under conditions and for a time sufficient for the cells to impregnate the material. Furthermore, the hardened PCA material may be prepared with a mold or as a composite with a leachable material (e.g., sugars, salt crystals, or enzyme-degradable fillers) to provide seeding chambers or areas within the device. In such approaches, the cells are preferably introduced into these chambers through a pipette or a syringe. Cells may also be introduced into the inventive hardened PCA material in vivo, by placing the material in the body adjacent to a source of desirable cells or cell precursors as described above for the hydrated precursor. In preferred embodiments, the hardened material is placed adjacent the periosteum or perichondrium, or is exposed to blood, fluids, or damaged host tissue that contains the desirable cells.

As those of ordinary skill will readily appreciate, the number of cells to be introduced into the inventive material (be it the hydrated precursor or the hardened PCA material) will vary based on the intended application of the seeded material and on the type of cell used. Where dividing autologous cells are being introduced by injection into the hydrated precursor, use of 20,000-1,000,000 cells per cm³are expected to result in cellular proliferation and extracellular matrix formation within the material. Where non-dividing cells are employed, larger numbers of cells will generally be required. In those cases where seeding is accomplished by host cell migration into the material in vivo, exposure of the material to fluids containing cells (e.g., bone-forming cells), or to tissue (e.g., bone) itself has proven to be effective to seed the material with cells without the need for inoculation with a specified number of cells.

Modification of Delivery Kinetics

One advantage of the PCA material present invention is that the rate of resorption of the material can be modulated through modifications in the preparative methods. Specifically, methods that lead to a more dense hardened product will generally result in a slower resorption time of the pure inventive PCA calcium phosphate in vivo. In this regard, there are a variety of ways to alter the density or resorption kinetics of the hardened product. These include adjustment of the volume of liquid used to create the paste, alteration of grain size of the starting materials, and compression of the paste during hardening. Composites, in which leachable or biodegradable particles or materials are incorporated into the paste, and ultimately the hardened PCA material, may also be prepared. The leachable or biodegradable materials may subsequently be removed (e.g., by leaching) from the hardened material in vivo, so that a highly porous implant is produced. Additionally, the inventive PCA material may be prepared with a distribution of densities within the same implant. One way this may be accomplished is by preparing in vitro-hardened PCA material of one density, pulverizing the hardened material to a desired grain size, and then mixing the pulverized material with a second PCA material paste designed to produce a different density PCA material. PCA materials made in this way will resorb asynchronously.

The use of overall smaller grain size material to prepare the PCA material precursor powder results in a longer time to resorb and/or reossify in vivo (see Examples 5 and 19). Since the ACP precursor is generally prepared at a very small grain size, when two components are used to produce the inventive vehicle, the grain size of the other non-ACP component is generally used to adjust resorption time. In this regard, the grain size may be adjusted by using a ground and sieved second component to select a specific grain size distribution for addition to the final mixture. In another embodiment, the second component is ground with the ACP for varying amounts of time to affect the resorption rate.

Composite materials with altered resorbability kinetics are produced by incorporating into the PCA material an "erosion rate modifier", which is a material whose presence alters the rate of resorbability of the device as a whole. Erosion rate modifiers that increase the rate at which the drug delivery device resorbs include any leachable or biodegradable compound that affects the solubility (e.g., by altering the porosity) of the device over time in vivo. Erosion rate modifiers that decrease the rate at which the drug delivery device resorbs include crystalline calcium phosphates, particularly hydroxyapatite, and diphosphate compounds.

Another way that the rate of resorption of the inventive PCA material can be modulated is through the action of osteoclast and/or macrophage cells. Osteoclasts, and possibly macrophages, naturally digest bone. According to the present invention, osteoclast or macrophage cells, or factors that modulate their development and/or activity, can be administered in conjunction with an inventive PCA material implant to accelerate or retard the rate of PCA material resorption.

For example, any agent that directly or indirectly (e.g., through osteoblasts) stimulates osteoclast activity or development may be employed to increase the resorption rate of a PCA material implant. Conversely, any agent that directly or indirectly inhibits osteoclast activity or development may be employed to reduce the resorption rate of an implant. Such stimulatory and inhibitory agents are well known in the art (see, for example, Athanasou, J. Bone Joint Surg., 78-A:1096, 1996 and Roodman Endocrine Rev. 17:308, 1996, each of which is incorporated herein by reference). For example, interleukin-1 (IL-1), colony stimulating factors (CSFs) such as macrophage (M)-CSF, transforming growth factor α (TGFα), tumor necrosis factor (TNF), interleukin 6 (IL-6), interleukin-11 (IL-11), interleukin-3 (IL-3), para-thyroid hormone (PTH), vitamin D3 metabolites (e.g., calcitriol), prostaglandins (under certain, known conditions), and oxygen free radicals are known to stimulate osteoclast development and/or activity. Where CSFs are utilized, subsequent administration of 1,25-dihydroxyvitamin D₃ can further stimulate osteoclasts; by contrast, concomitant administration of colony stimulating factors and 1,25-dihydroxyvitamin D₃inhibits osteoclast.

Other factors that inhibit osteoclast development and/or activity include transforming growth factor-β (TGFβ), γ-interferon, interleukin-4 (IL-4), nitric oxide, antibodies, for example, against the osteoclast vitronectin receptor, calcitonin, and prostaglandins (under certain, known conditions).

Of course, it is also possible to introduce osteoclasts themselves (or osteoclast precursor cells, preferably in combination with agents that stimulate their differentiation into osteoclasts) into a PCA material implant in order to stimulate its resorption.

Agents that alter PCA material resorption rate may be administered systemically or locally. Local administration is preferably accomplished by introducing the agent into, or associating the agent with, the material itself, preferably according to the procedures described herein. Where local administration is being employed, it is preferred that diffusion of the agent away from the PCA material implant be minimized. For example, relatively insoluble agents are preferred because it is less likely that they will diffuse away from the implant and exert undesirable effects on other cells within the body.

Applications

As alluded to above, the cell seeded PCA material of the present invention can be usefully employed in any of a variety of in vivo and in vitro systems. For example, the material may be used to deliver biologically active agents or cells to any of a variety of sites in a body (preferably a human body, though veterinary applications are also within the scope of the invention. Alternatively or additionally, the material may be used in bone tissue or repair applications or augmentation plastic therapy in vivo. The material may also be employed as a cell encapsulation membrane or matrix, or in artificial organ construction or repair.

In vitro, the material may be used as a three dimensional cell culture matrix, and as a model for analyzing osteoclast, osteoblast, chondrocyte, and/or macrophage cultures, progenitor cell differentiation, and/or reossification and calcium phosphate resorption. The material is particularly useful for tissue formation and/or degradation studies, for example employing cells such as progenitor cells, stem cells, osteocytes, osteoclasts, osteoblasts, chondrocytes, macrophages, myoblasts, and fibroblasts. The material may also be employed to accomplish in vitro delivery of a biologically active agent.

Certain preferred applications are discussed in more detail below, but the discussion is intended only for purposes of exemplification and is not intended to be limiting.

When used as an in vivo or in vitro delivery vehicle, the PCA material of the present invention offers the advantage of controlled, localized delivery. As is well known, smaller amounts of biologically active agent are required when the agent is delivered to a specific site rather than administered systemically. Furthermore, potential toxic side effects of the agent are minimized when the agent is delivered from the delivery vehicle of the present invention. Also, the agent's activity is maximized because it is protected within the delivery vehicle until it is delivered to its site.

The PCA material of the present invention can be injected or implanted into any acceptable tissue. Oral formulations are also considered within the scope of the invention. Preferred delivery sites include sites in bone, muscle, the spinal cord, the central nervous system, the interperitoneal cavity, subcutaneous locations, and the vitreous and aqueous humor of the eye. When the PCA material is delivered to a site under circumstances where implant migration is a concern, anchoring sutures or hooks may be incorporated into the vehicle so that it can be attached and maintained in position. When appropriate, the PCA material may be anchored by insertion into a bony site (see below). Particular applications and preferred delivery sites are discussed in more detail below:

Delivery of Biologically Active Agents to Bony Sites

The PCA material of the present invention has particular advantages for delivery of biologically active agents to sites in bone. Implantation of a delivery vehicle formed from PCA material of the present invention in a bony site may alternatively or additionally be utilized to anchor a delivery vehicle and accomplish systemic drug delivery, or may be utilized to accomplish delivery to a site adjacent to, though not strictly speaking "within", the bone. FIG. 9 depicts many useful applications of the PCA material of the present invention in bony sites.

Naturally-occurring bone mineral is made of nanometer-sized, poorly-crystalline calcium phosphate with apatitic structure. However, unlike the ideal stoichiometric crystalline hydroxyapatite, Ca₁₀(PO₄)₆(OH)₂, with atomic Ca/P ratio of 1.67, the composition of bone mineral is significantly different and may be represented by the following formulae,

Bone mineral non-stoichiometry is primarily due to the presence of divalent ions, such as CO₃^2-and HPO₄^2-, which are substituted for the trivalent PO₄^3-ions. Substitution by HPO₄^2-and CO₃^2- ions produces a change of the Ca/P ratio, resulting in Ca/P ratio which may vary between 1.50 to 1.70, depending on the age and bony site. Generally, the Ca/P ratio increases during aging of bone, suggesting that the amount of carbonate species typically increases for older bones. It is the Ca/P ratio in conjunction with nanocrystalline size and the poorly-crystalline nature that yields specific solubility property of the bone minerals. And because bone tissues undergo constant tissue repair regulated by the mineral-resorbing cells (osteoclasts) and mineral-producing cells (osteoblasts), solubility behavior of minerals is important in maintaining a delicate metabolic balance between these cells activities.

The PCA material of the present invention is a nano-size, poorly crystalline solid with a Ca/P ratio comparable to that of natural bone minerals. The material is bioresorbable, can be produced at low temperatures, and is readily formable and injectable. For all of these reasons, the inventive material is particularly well suited for drug delivery in bony sites. Furthermore, this synthetic PCA material can support bone growth so that it is eventually replaced by the patient's own bone. It should be borne in mind, however, that bone ingrowth may well affect the resorbability rate of the drug delivery material of the present invention. Accordingly, it may be desirable in certain circumstances (e.g., where the biologically active agent must be delivered according to a precise, predetermined administrative schedule) to reduce bone growth into the drug delivery vehicle, for example by blocking penetration of osteocytic or chondrocytic cells or precursors. In most circumstances, ossification can be avoided by placing the device at some distance away from bone. Generally, 1 mm will be sufficient, although greater distances are preferred. Also, compounds such as Indian hedgehog gene and gene products, parathyroid hormone-related protein (PTHRP) and PTHRP receptor agonists may be included in, on, or adjacent to the drug delivery device in order prevent bone growth.

In other circumstances, such bone ingrowth can desirably be encouraged. As shown in Examples 14, 17, and 18, the PCA calcium phosphate material can be placed into bony sites and allowed to resorb in a manner that results in its apparent complete (100%) replacement with new bone. Where optimal ossification is desired, the devices and objects may be seeded with bone forming cells (see below). This goal is most easily accomplished by placing the device in contact with a source of the patient's own bone forming cells. Such cells may be found in bone tissue or in bone-associated blood or fluids, including exogenous fluids which have been in contact with bone or bone materials or regions, including the periosteum, cancellous bone or marrow. In the case of devices such as screws and pins, the introduction of which into bone is accompanied by bleeding, no further seeding is required. For plates, which oppose only cortical bone, induction of a periosteal lesion which will contact the device is recommended. In yet other embodiments, it will be useful to surgically prepare a seating within the bone by removing a portion of cortical bone at the implant site. Other steps may also be taken to augment ossification, including introduction bone forming cells harvested from the patient into the graft, or incorporation of trophic factors or bone growth inducing proteins into, or onto the device. Non-autologous bone cells are also within the scope of the invention if the desired amount of bone regeneration occurs prior to host rejection of the bone forming cells. In this regard, immunosuppressants may be administered to the device recipient, in some cases by incorporation into the device. Thus, cells or tissues obtained from primary sources, cell lines or cell banks may all be useful in certain embodiments.

Certain categories of biologically active agents are expected to be particularly suitable for delivery to bony sites. For example, where the drug delivery vehicle is applied to a damaged bone site, it may be desirable to incorporate bone regenerative proteins (BRPs) into the vehicle. BRPs have been demonstrated to increase the rate of bone growth and to accelerate bone healing (see, for example, Appel et al., Exp. Opin. Ther. Patents 4:1461, 1994). Exemplary BRPs include, but are in no way limited to, Transforming Growth Factor-Beta (TGF-β), Cell-Attachment Factors (CAFs), Endothelial Growth Factors (EGFs), OP-1, and Bone Morphogenetic Proteins (BMPs). Such BRPs are currently being developed by Genetics Institute, Cambridge, Mass.; Genentech, Palo Alto, Calif.; and Creative Biomolecules, Hopkinton, Mass. Bone regenerative proteins and trophic factors can also be used to stimulate ectopic bone formation if desired. The inventive PCA material containing BMP-7 can be placed subcutaneously, and bone formation will occur within 1-2 months.

Antibiotics and antiseptics are also desirably delivered to bony sites using the PCA drug delivery vehicle of the present invention. For example, one of the major clinical implications arising from bone-graft surgery is a need to control the post-operative inflammation or infection, particularly infection associated with osteomyelitis. An embodiment drug delivery device of the present invention, including an antibiotic, could be used as (or in conjunction with) an improved bone graft to reduce the chances of local infection at the surgery site, contributing to infection-free, thus faster, bone healing process. The efficacy of antibiotics is further enhanced by controlling the resorption of the poorly crystalline hydroxyapatite such that it dissolves at a rate that delivers antibiotic peptides or its active component at the most effective dosage to the tissue repair site.

Exemplary antibiotics include, but are in no way limited to, penicillin, tetracycline, kanamycin, gentamycin, chlortetracycline hydrochloride (aureomycin), minocyline, dosycycline, vanomycin, bacitracin, neomycin, erythromycin, streptomyan, cephalosporins, chloramphenicol, oxytetracycline (terramycine), and derivatives thereof. Antibiotics and bone regenerating proteins may be incorporated together into the PCA material of the present invention, to locally deliver most or all of the components necessary to facilitate optimum conditions for bone tissue repair.

Other biologically active agents that are desirably delivered to bony sites include anti-cancer agents, for example for treatment of bone tumors (see, for example, Otsuka et al., J. Pharm. Sci. 84:733, 1995). The drug delivery vehicle of the present invention is particularly useful, for example, where a patient has had a bone tumor surgically removed because the synthetic, PCA material of the present invention can improve the mechanical integrity of the bone site while also treating any remaining cancer cells to avoid metastasis. Exemplary anti-cancer agents include, for example, methotrexate, cisplatin, prednisone, hydroxyprogesterone, medroxyprogesterone acetate, megestrol acetate, diethylstilbestrol, testosterone propionate, fluoxymesterone, vinblastine, vincristine, vindesine, daunorubicin, doxorubicin, hydroxyurea, procarbazine, aminoglutethimide, mechlorethamine, cyclophosphamide, melphalan, uracil mustard, chlorambucil, busuflan, carmustine, lomusline, dacarbazine (DTIC: dimethyltriazenomidazole carboxamide), fluorouracil, 5-fluorouracil, cytarabine, cytosine arabinoxide, mercaptopurine, 6-mercaptopurine, thioguanine.

Additional biologically active agents that can desirably be incorporated into the synthetic PCA drug delivery system of the present invention for delivery to bony sites are agents that relieve osteoporosis. For example, amidated salmon calcitonin has been demonstrated to be effective against osteoporosis.

Vitamin D and Vitamin K are also desirably delivered to bony sites, as are angiogenic factors such as veg f, which can be used when it is desirable to increase vascularization.

Bone Production and Healing

In preferred embodiments of the present invention, the PCA material is seeded with bone-forming cells or precursors thereof. Preferably, the PCA material is formulated, and the cell population is selected, so that the PCA material becomes ossified within a period of about 4-12 weeks.

In particularly preferred embodiments of the invention, the seeding is accomplished by placing the PCA material in contact with a source of the host's own bone-producing cells. Such cells are found in bone tissue or in bone-associated blood or fluids, including exogenous fluids that have been in contact with bone (including cancellous bone), bone materials, or bone regions such as the periosteum or the marrow.

Various modes of introducing the PCA material of the invention into bony sites are thoroughly described in U.S. application entitled "Orthopedic and Dental Ceramic Implants" and filed on even date herewith. Where the PCA material is to be implanted into a bony site in vivo in a manner that induces bleeding, such bleeding can effectively introduce bone-forming cells into the material so that no further seeding is required. Approaches that induce bleeding include those in which the PCA material is formed into a screw or pin, or is applied in conjunction with a screw or pin made from another material.

Where the PCA material is used as or in conjunction with a plate that opposes only cortical bone, it is preferred that a periosteal lesion be introduced in a manner that creates contact between the PCA material and the lesion, so that cells may penetrate into the PCA material from the lesion. Similarly, in some embodiments of the invention, it will be useful to surgically prepare a PCA device seating within the bone by removing a portion of cortical bone at the implant site. Cells at the implant site will migrate into and seed the PCA material.

Of course, it is not required that the PCA material devices be seeded by in vivo impregnation of the host's own cells. Bone forming cells harvested from the host may be introduced in vitro into the device, so that a seeded composition is implanted in the host. Furthermore, seeding with non-autologous bone cells is also within the scope of the invention, but care must be taken to ensure that a desired amount of bone growth occurs prior to host rejection of the bone forming cells. Such non-autologous cells can be obtained from any of a variety of sources, including but not limited to primary sources, cell lines, and cell banks.

Bone formation in and around the PCA material can be enhanced by the incorporation of trophic factors and/or bone-growth inducing factors into, or onto, the PCA material device.

Osseous Augmentation

Seeded PCA compositions of the present invention are useful for the enhancement or alteration of the shape of bony structures (e.g., a chin). For such applications, the PCA material may be supplied either as a pre-hardened shape or a molded putty form and applied to a bony surface. Generally, PCA material formulations selected for augmentation applications will be those that resorb on a relatively slower time course, typically requiring 6-12 weeks for resorption.

PCA material employed in augmentation applications are typically seeded through application of cells or cell lines to the PCA material, although some preferred embodiments involve host cell seeding. The term "host cell seeding" encompasses any method by which cells of the host are introduced into the PCA material. For example, the term encompasses migration of host cells into a device implanted in vivo, as well as assisted migration accomplished by placing bone blood or fragments of the periosteum on or in contact with the device (in vivo or in vitro), among other things.

Cartilage Production and Healing

Damage to cartilage can result in serious physical deformations. Currently, the most common treatment for loss of cartilage is replacement with a prosthetic material, but many difficulties have been encountered with this approach. As put by one of the leaders in the field. "The lack of truly biocompatible, functional prostheses can have profound and tragic effects for those individuals who have lost noses or ears due to burns or trauma". Seeded PCA compositions of the present invention offer an attractive alternative in which the PCA material acts as a formable scaffold into and within which tissue can grow. The PCA material is bioresorbable so that, eventually, the PCA material implant can be replaced with natural tissue; the negative effects of long-term prosthetic implants can therefore be avoided.

The PCA material of the present invention can be seeded with cartilage-forming cells in order to optimize chondrogenesis. Preferably, this seeding is accomplished by placing the device in contact with a source of the host's own cartilage-forming cells (e.g., chondrocytes) or precursors thereto. Such cells are found in cartilage-associated blood or fluids, including exogenous fluids that have been in contact with cartilage or cartilagenous materials. Thus, fluids that have been in contact with the perichondrium, cartilage, or marrow typically contain such cells.

In many cases, e.g., a PCA material device designed for augmentation of a damaged ear, seeding can be accomplished by placing the PCA device in contact with the breached region of the perichondrium. In other cases, it will be useful to surgically prepare a seating for the PCA device within existing cartilagenous tissue by removing a portion of the cartilage at the implant site.

In some embodiments of the present invention, additional steps may be taken to augment chondrogenesis associated with the seeded PCA material. For example, cartilage-forming cells harvested from the patient may be introduced into the device in addition (or as an alternative to) cells that impregnate it after implantation in vivo. Alternatively or additionally, trophic factors or cartilage growth-inducing factors may be incorporated into or onto the device.

It should be clear that autologous cells are not required for the seeded PCA compositions employed in cartilage-forming applications; non-autologous cells are also within the scope of the invention so long as the cells are selected and the PCA material is formulated so that a desired amount of cartilage regeneration occurs prior to host rejection of the cartilage-forming cells. Thus, cells or tissues obtained from primary sources, cells lines, or cell banks are useful in the practice of this embodiment of the present invention.

Ectopic Bone or Cartilage Production

The seeded PCA material compositions of the present invention can be used to produce bone or cartilage formation at a site at which bone or cartilage does not normally occur. Introduction of a PCA composition into which bone- or cartilage-producing cells have been seeded into an in vivo implant site will result in bone or cartilage formation at that site. In preferred embodiments, the PCA material contains growth and/or trophic factors in addition to the seeded cells, so that maintenance of the ectopically-formed bone or cartilage can be prolonged. Once it has been produced, such ectopic tissue may either be left in place or may be surgically removed, depending on its intended use. Alternatively or additionally, trophic or growth factors external to the implant may be provided, e.g., through the use of encapsulated cells, polymer implants, or other method of factor delivery (see, for example, Aebischer et al., U.S. Pat. No. 4,892,538; Sefton, U.S. Pat. No. 4,353,888 and Winn et al. Experimental Neurology 140:126 (1996)).

Ectopic tissues may be formed in vitro using inventive seeded PCA material compositions. Preferably, a hydrated precursor is prepared, is shaped by hand or through the use of a mold or form, and is subsequently hardened at an elevated temperature (27-50° C.). Alternatively, the PCA material may first be hardened and subsequently be machined or otherwise formed into a desired shape. Cell seeding can be accomplished by any of the methods described herein, so that ectopic tissue will be formed in vitro in the desired shape. Generally, to ensure that the shape is maintained during cell growth, it will be desirable to inhibit the action of degredative enzymes and cells, as is known in the art.

Cell Encapsulation Matrix

The PCA material of the present invention provides an excellent growth matrix for use within the cell encapsulation environment. Use of this material can prevent cell settling, provide cell dispersion, and optimize nutrient localization by encapsulated cells. Thus, according to the invention, cells may be encapsulated within encapsulation devices in the presence of the hydrated precursor or hardened PCA of the present invention, and the resultant encapsulated devices may then be implanted in vivo for use in encapsulated cell therapy applications. Useful techniques for preparing and using cell encapsulation devices are described in, for example, Winn et al., Expt. Neurol. 140:126, 1996 and Aebischer, U.S. Pat. No. 4,892,538; Sefton, U.S. Pat. No. 4,353,888, and Kordower et al., Cell Transplantation, 14:155, 1995, each of which is incorporated herein by reference.

Research Applications

The PCA material of the present invention, due to its ease of preparation, mild formation conditions, sparing solubility in most aqueous systems, and tractability for use in cell-embedding applications, provides an attractive three-dimensional growth matrix for use in research and production tissue culture applications. Furthermore, the material is useful for tissue formation and/or degradation studies (e.g., of bone or cartilage). Preferably, the material employed in such studies in seeded with cells such as (but not limited to) progenitor cells, stem cells, osteocytes, osteoclasts, osteoblasts, chondrocytes, macrophages, myoblasts, and fibroblasts.

Diagnostics

Cell-seeded PCA materials of the present invention may be employed in diagnostics that detect various health or disease states. For example, the inventive PCA material can be used in qualitative or quantitative assays to determine the bone- or cartilage-forming potential of cells taken from a patient to be diagnosed. The inventive material can also be used in diagnostics to assay vascularization and hard tissue degradation. Various soft tissue diagnostics are also made possible with the inventive PCA material compositions.

Delivery of Biologically Active Agents to Subcutaneous Implant Sites

Application of the present drug delivery device is not limited to bony sites, of course. In non-bony sites, the device is known resorb without ossification.

Placement of the instant delivery device subcutaneously is particularly useful for more systemic administration of biologically active compounds. The administration of estrogens and/or progesterones for the used in fertility control is an example of a subcutaneous application. Additionally, the administration of antigens and/or vaccines may be accomplished through subcutaneous implantation.

Delivery of Biologically Active Agents to Central Nervous System

The delivery of therapeutic substances to the central nervous system may be accomplished with the inventive delivery vehicles. Useful therapeutic substances include the delivery of γ-aminobutyric acid to epileptic foci, the delivery of L-dopa or dopamine in the striatum or substantia nigra for the treatment of Parkinson's disease, the delivery of growth factors for the prevention of neural degeneration such as GDNF in the lateral ventricles, striatum or substantia nigra for the treatment of Parkinson's disease, the administration of NGF to cortical and other regions for the treatment of Alzheimer's disease, or the administration of CNTF to the sacral or lumbar spinal cord for the treatment of amyelolateral sclerosis.

Other: Delivery of Biologically Active Agents to Sites

Other potential delivery sites include intramuscular, interperitoneal, and occular areas.

Claim 1 of 18 Claims

1. A bioresorbable implant composition comprising:

a calcium phosphate;

a first agent that directly or indirectly stimulates osteoclast activity, wherein said first agent modulates the resorption of the calcium phosphate at an implant site; and

a second agent that is biologically active, wherein said first and second agents are different.

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