Physically stable composition and method of use thereof for osseous repair.

Cross-Reference to Related Application

This application is a Continuation-In-Part of U.S. application Serial No. 811,677, filed December 20, 1985.

Background of the Invention

Currently, the most effective method for treatment of severe or nonhealing osseous defects is autogenic bone grafting, which involves the transplan­tation of bone from another part of a patient's body into the defect. Significant disadvantages are asso­ciated with autogenic bone grafting, including donor site morbidity and limited tissue availability. Bone banks have been established to provide an alternative source of bone grafting material, consisting of allo­genic freeze-dried bone. Allogenic bone grafts, how­ever, are very expensive and do not heal as well as do fresh autogenic bone grafts.

Attempts to overcome these problems have involved the use of calcium phosphates and apatites. They have been evaluated as either dense or porous implants, with various types and sizes of porosity, including replicas of marine coral. Hydroxylapatite implants are generally considered to be somewhat more stable <u style="single">in</u><u style="single">vivo</u> than tricalcium phosphate implants, which show a greater tendency to be absorbed. These ceramic materials are generally implanted in particu­late form to fill bone defects, which can present handling difficulties.

Hydroxylapatite and tricalcium phosphate par­ticles both apparently provide a useful scaffold for bone ingrowth into a bone defect, but these ceramics do not have any inductive or accelerating effect on bone repair. A significant problem with implants of calcium phosphate mineral particles relates to keeping the par­ticles at the implantation site. Various approaches have been used to try to inhibit migration of the mineral particles from the implantation site, including adding the particles to a collagen matrix and mixing the particles with plaster of paris (See European patent application 197,693, and U.S. Patent No. 4,619,655, respectively).

Demineralized, lyophilized bone has also been used as a component of osteoinductive agents. See M. R. Urist, <u style="single">Science</u>, <u style="single">150</u>, 893 (1965). Recently, M. R. Urist et al., in U.S. Pat. No. 4,294,753 and in <u style="single">Proc. Natl. Acad. Sci. USA</u>, <u style="single">76</u>, 1828 (1979), have disclosed the use of proteins such as "protein osteoinductive factor" (OF) derived from such bone matrices with various carriers to induce new bone formation. However, it can be difficult to reproducibly prepare these materials, which must be characterized by various bioassay systems. See, for example, R. Nathan et al., U.S. Patent No. 4,563,350.

Sauk and Van Kampen disclose compositions which are effective to promote new bone formation upon introduction of the composition into osseous defects in U.S. patent application Serial No. 811,677, filed December 20, 1985, the disclosure of which is incor­porated by reference herein. Preferred compositions are prepared by mixing a phosphophoryn calcium salt and type I collagen in a ratio of the salt to the collagen of about 3.0-0.1:1 to yield a solid composition. These compositions have the appearance and texture of soft sponges. The low mechanical strength and highly com­pressible nature of these sponges can limit their application in the treatment of segmental defects.

Therefore, a need exists for physically stable compositions useful to repair osseous defects which can be readily manipulated during formulation and implantation. A further need exists for compositions useful for osseous repair which can be prepared repro­ducibly, e.g., which incorporate well characterized components.

Brief Description of the Invention

The present invention is directed to a solid composition which is prepared by a process comprising mixing a phosphophoryn calcium salt and type I collagen in a salt:collagen weight ratio of about 3.0-0.1:1, preferably about 2.5-0.5:1, with a large amount of a particulate polycrystalline calcium phosphate ceramic to yield a solid matrix. The particulate ceramic is employed in an amount which is effective to substan­tially improve the physical stability of the matrix of the resultant porous solid. The resultant spongy solid is resilient but is substantially less compressible than the sponges disclosed in Serial No. 811,677. Therefore, it is easier to predetermine the amount of the present composition which will suffice to complete­ly fill a given defect void. Furthermore, the present compositions are more rigid, and thus, provide more tactile feedback to the surgeon or dentist performing the implant. Thus, they can be more easily applied to and positioned in the defect.

The present invention is also directed to the use of these compositions to promote bone formation upon their introduction into osseous defects. It is believed that the particulate calcium phosphate cera­mics will enhance the long-term repair and remodeling of new bone in osseous defects over that obtainable with implant compositions consisting essentially of phosphophoryn calcium or phosphophoryn calcium-­collagen.

Therefore, the compositions of the present invention are useful as bone graft substitutes in the repair of osseous defects and to promote osseous forma­tion. For example, they are useful to fill defects created during the treatment of osteosarcomas or bone cysts, to promote the repair of nonunions and to repair alveolar clefts. The present compositions are at least partially absorbed in a short period after initiating a cascade of events which leads to their complete replacement by new bone. The compositions of the pre­sent invention are particularly useful for the treat­ment of large osseous defects, where there is a requirement for a rapid diminishment of an osseous void in order to re-establish the cortical plate and reduce the possibility of fracture. Furthermore, the present compositions are well-characterized and can be obtained reproducibly.

Detailed Description of the Invention

According to the present invention, new bone formation can be promoted <u style="single">in</u><u style="single">vivo</u> in osseous defects by introduction thereinto of a composition comprising phosphophoryn calcium. To facilitate manipulation by the physician and to provide a structure to direct the deposition of new bone, these compositions preferably comprise a mixture of phosphophoryn calcium, a matrix material such as type I collagen and a particulate calcium phosphate ceramic. These compositions are intended to facilitate matrix-mediated mineralization, whereby the collagen defines a structural matrix and the phosphophoryn salt regulates and directs mineral deposition in terms of its location, crystallinity and association with the calcium phosphate ceramic par­ticles. The calcium phosphate particles also interact with the collagen to improve the physical properties of the collagen matrix by reducing its compressibility and increasing its mechanical strength.

Thus, the present compositions do not simply provide a static physical "scaffold" for bone ingrowth, but are believed to create a dynamic ionic front that accelerates new bone formation by mimicking the miner­alization front created by dentin <u style="single">in</u><u style="single">vivo</u>. Another advantage of the invention is that the compositions are able to be partially absorbed after initiating a cascade of events that leads to a more rapid and complete replacement of osseous voids than can be accounted for by normal wound healing processes.

Phosphophoryn Salt

Phosphophoryns are acidic, phosphorous-­containing proteins which can be isolated from the non-­collagenous proteins which are constituents of the organic extracellular matrix of certain mineralized tissues. <u style="single">In</u><u style="single">vivo</u>, these proteins may act to initiate the calcification of dentin by chelating calcium ions. Purified dentinal phosphophoryn has a high affinity for calcium ion (Ca⁺²). Its binding sites exhibit dissoci­ation constants (Kd) of 1.3 x 10⁻⁷M and 0.85 x 10⁻⁵M. However, sodium, potassium and magnesium ions can effectively compete with Ca⁺² for these binding sites. The calcium salts of phosphophoryn employed in the pre­sent invention may reduce this deleterious competitive binding.

Conventional methods for the purification of the preferred dentinal phosphophoryn employ precipita­tion of decalcified dentin with calcium salts. Follow­ing purification, these salts can be employed as the dentinal phosphophoryn calcium component in the present compositions. The purification of dentinal phosphopho­ryn has been described by W. T. Butler et al., <u style="single">Collagen Rel. Res.</u>, <u style="single">1</u>, 187 (1981) and by D. Cocking-Johnson et al., in <u style="single">Collagen Rel. Res</u>., <u style="single">3</u>, 505 (1983), the disclo­sures of which are incorporated by reference herein.

Synthetic phosphophoryns may be produced in the laboratory by recombinant DNA techniques. Alterna­tively, phosphophoryn analogs useful in the present invention may be chemically synthesized by conventional reactions employed in polypeptide synthesis.

Collagen

Collagen is the general designation of a group of at least four connective tissue proteins which are designated as types I-IV. Type I collagen accounts for a large part of the organic mass of bone, and has also been isolated from intervertebral disc, liver, tendon and kidney tissue, and in combination with type III collagen from skin, sclera and spleen. The preci­pitational behavior of native pepsin-resistant collagen molecules at relatively low ionic strength has been used extensively for their purification from other pro­teins. In addition, differential solubilities have been observed for types I, II and III collagens which can be fractionated from one another by precipitation at different salt concentrations at neutral pH. For example, at acidic pHs, both type I collagen and type III collagen derived from skin precipitate at 0.7-0.9M NaCl concentration, while at neutral pH, type I preci­pitates at 2.6M NaCl while type III precipitates in 1.5-1.7M NaCl. See E. H. Epstein, Jr., <u style="single">Biol. Chem</u>., <u style="single">249</u>, 3225 (1974).

Due to the difficulties encountered in pre­paring pure collagen of a given type, the term "type I collagen" as used herein is intended to refer to pure type I collagen and any collagen preparation which comprises a substantial proportion of type I collagen, preferably at least a major proportion of type I colla­gen.

Calcium Phosphate Ceramic

Polycrystalline calcium phosphate ceramics useful in the present compositions are generally formed by the high temperature sintering of various calcium phosphate minerals. These minerals are found commonly in nature and also can be prepared synthetically by well-known solution precipitation techniques. A par­ticular calcium phosphate, namely tribasic calcium phosphate (hydroxylapatite), is the mineral component of bones and teeth. Therefore, calcium phosphate-based ceramics have been evaluated as hard tissue implants. The most widely investigated polycrystalline calcium phosphate ceramic implants employ tribasic calcium phosphate (hydroxylapatite, [Ca₁₀(PO₄)₆(OH)₂]), or <u style="single">beta</u>-­tricalcium phosphate [Ca₃(PO₄)₂]. The preparation and properties of these and other useful ceramic materials is discussed in a review article by M. Jarcho, in <u style="single">Clinical Orthopaedics and Related Research</u>, <u style="single">157</u>, 259 (1981), the disclosure of which is incorporated by reference herein.

The choice of any given particulate calcium phosphate ceramic will be directed by (a) the particle size, (b) the particle density, (c) the desired matrix density, and (d) the desirability of particle absorp­tion. For example, the preferred polycrystalline calcium phosphate ceramics for use in the present com­positions consist of small particles of dense hydroxyl­apatite (ca. 1-10 microns in diameter), which are readily supported by relatively low density (ca. 0.5-1.5 mg/cc) phosphophoryn calcium-collagen matrices. Larger particles of dense hydroxylapatite of a size greater than about 100 microns in diameter can be supported by a more dense (ca. 2.5-7.5 mg/cc) phospho­phoryn calcium-collagen matrix. Because the specific gravity of dense tricalcium phosphate is approximately the same as that of hydroxylapatite, it follows that tricalcium phosphate particles of similar size can be substituted for hydroxylapatite. Mixtures of these two materials can also be used. It may be possible to use larger-sized particles of porous calcium phosphate minerals in the lower density phosphophoryn calcium-­collagen matrix because the lower density of the porous particles should allow larger particles to be supported by the matrix.

Preparation

The compositions of the present invention can be prepared by adding the desired amount of the phos­phophoryn calcium salt as a powder to an aqueous solu­tion containing the desired amount of collagen. The powdered calcium phosphate ceramic is then added, and the water is removed <u style="single">in</u><u style="single">vacuo</u>, e.g., by lyophilization. The resultant material is a tan, hardened sponge which can be directly implanted into osseous defects without further purification.

The preferred compositions are prepared by mixing the particulate calcium phosphate ceramic, phos­phophoryn calcium, and type I collagen in a weight ratio of about 775-15:3-0.1:1, preferably about 400-20:3-0.1:1, most preferably about 200-35:2.5-0.5:1. One particularly preferred embodiment employs a weight ratio of hydroxylapatite:dentinal phosphophoryn calcium:type I collagen of about 90:1:1.

The invention will be further described by reference to the following detailed examples.

Example I--Physically Stable Osseous Repair Compositions

1. Materials

A. Dentinal Phosphophoryn Calcium Salts

Small pieces of cleaned bovine dentin were stirred for 18 hr at 4°C in 4M guanidine-HCl containing four protease inhibitors [1 mM iodoacetate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mg/ml soy­bean trypsin inhibitor and 5 mM n-ethylmaleimide]. The dentin was rinsed thoroughly with water, placed in dialysis bags and decalcified by dialysis against 0.25M EDTA containing the protease inhibi­tors at 4°C. Decalcification required a minimum of 3 changes of EDTA, each exposure lasting at least 72 hours. The supernatant inside the dialysis bags was isolated and dialyzed against distilled water for at least 4 days. The phosphophoryns were then precipitated with calcium chloride as described by Y. Kuboki et al., <u style="single">J. Dent. Res</u>., <u style="single">58</u>, 1926 (1979) and the mixture was stirred for 18 hr at 25°C. The dentinal phosphophoryn calcium salts were isolated by centrifugation, washed with 1.0M aqueous CaCl₂ and dried by lyophilization.

B. Unenriched Bovine Skin Collagen

Bovine skin was digested with 1 mg/ml pepsin and the crude product precipitated with NaCl (0.7M). The collagen was then dialyzed extensively against 0.5M acetic acid and lyophilized. The supernatant was treated with NaCl (1.5M) to yield a precipitate which was dialyzed extensively against 0.5M acetic acid and lyophilized to yield the product.

C. Type I and Type III Collagen

Collagen was prepared from bovine skin by the pro­cedure of Uitto as described in <u style="single">Arch. Biochem. and Biophys</u>., 371-379 (1979). Accordingly, samples of calf skin were dissected and the subcutaneous tissues removed. The specimens were then rinsed with cold (+4°C) 0.15M NaCl-0.05M Tris-HCl, pH 7.5. The skin was then minced extensively with scissors in 0.5M acetic acid. Pepsin (Worthington Diagnos­tics, Freehold, NJ, 2X crystallized) was added to a final concentration of 0.1 mg/ml, and the samples were incubated at 4°C for 16 hr. At the end of the incubation period, the homogenate was centrifuged at 30,000 g for 60 min at 4°C. The pellet was then dissolved and adjusted to pH 8.5 with cold 1.0M NaOH, and extensively dialyzed against 0.4M NaCl-0.1M Tris-HCl, pH 7.5.

In order to separate type I collagen, the solubil­ized material was fractionated by the sequential precipitations with 1.5 and 2.5M NaCl. After the slow addition of solid NaCl to any given concentra­tions, the samples were stirred for 24 hr at 4°C and then centrifuged at 30,000 g for 60 minutes.

The pellet resulting from precipitation at 2.5M NaCl was dissolved in and dialyzed against 0.5M acetic acid and then lyophilized. The type I-­enriched collagen appears as a soft white spongy substance.

The pellet resulting from the precipitation with 1.5M NaCl at neutral pH consists of a 1:1 distribu­tion of type I and type III collagen. See J. Uttio, <u style="single">Arch. Biochem. Biophys</u>., <u style="single">192</u>, 371 (1979).

D. Osseous Repair Compositions

Bovine type I collagen was dissolved in 0.5M acetic acid at 4°C for 24 hr with constant mixing. The resulting solutions were then centrifuged at 30,000 g for 30 min and the supernatant collected. The concentration of the collagen was adjusted to 1 mg/ml by measuring the absorption at 247nm for which a standard curve has been created based on absorption and hydroxyproline ratios. To these latter collagen solutions, 1 mg/ml of phosphophoryn calcium was added followed by particulate hydroxyl­apatite in the vol. (phosphophoryn calcium-collagen solution):vol. (mineral) ratios summarized on Table 1, below. <tables id="tabl0001" num="0001"><table frame="all"><title>Table 1</title><tgroup cols="3" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="52.50mm" /><colspec colnum="2" colname="col2" colwidth="52.50mm" /><colspec colnum="3" colname="col3" colwidth="52.50mm" /><thead valign="top"><row><entry namest="col1" nameend="col1" align="center"><u style="single">Example</u></entry><entry namest="col2" nameend="col2" align="center"><u style="single">Volume Phosphophoryn Ca⁺²-Collagen Mixture*</u></entry><entry namest="col3" nameend="col3" align="center"><u style="single">Volume Hydroxylapatite Powder**</u></entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">A</entry><entry namest="col2" nameend="col2" align="right">1</entry><entry namest="col3" nameend="col3" align="char" char=".">1.0</entry></row><row><entry namest="col1" nameend="col1" align="left">B</entry><entry namest="col2" nameend="col2" align="right">1</entry><entry namest="col3" nameend="col3" align="char" char=".">0.5</entry></row><row><entry namest="col1" nameend="col1" align="left">C</entry><entry namest="col2" nameend="col2" align="right">1</entry><entry namest="col3" nameend="col3" align="char" char=".">0.25</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left">D</entry><entry namest="col2" nameend="col2" align="right">1</entry><entry namest="col3" nameend="col3" align="char" char=".">0.1</entry></row></tbody></tgroup><tgroup cols="3" colsep="0" rowsep="0"><colspec colnum="1" colname="col1" colwidth="52.50mm" /><colspec colnum="2" colname="col2" colwidth="52.50mm" /><colspec colnum="3" colname="col3" colwidth="52.50mm" /><tbody valign="top"><row><entry namest="col1" nameend="col3" align="justify">* 1 mg/ml phosphophoryn-Ca⁺² and 1 mg/ml type I collagen in 0.5M acetic acid.</entry></row><row><entry namest="col1" nameend="col3" align="justify">** 1-10 micron diameter particles (361 mg/cc)</entry></row></tbody></tgroup></table></tables>

The range of hydroxylapatite mineral to aqueous phosphophoryn-collagen mixture that possessed the most desired qualities was 0.5-0.1:1 (vol:vol.) This represents a weight ratio of mineral:phosphophoryn calcium:collagen of 180-35:1:1. The most preferred quality of materials was achieved at a ratio of calcium phosphate mineral to aqueous phosphophoryn-collagen mixture of 1:0.25 vol:vol. This is a weight ratio of mineral:phosphophoryn calcium:collagen of 90:1:1.

All of the compositions prepared with a starting collagen solution concentration of 1 mg/cc and with fine hydroxylapatite mineral particles of about 1-10 microns in size possessed the hardened, sponge-­like characteristics desired for an osseous replacement matrix. Samples prepared with larger particles of hydroxylapatite mineral (759 mg/cc) required a starting collagen solution concentration of 5 mg/cc to support the mineral particles in the matrix.

The invention has been described with refer­ence to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remain­ing within the spirit and scope of the invention.