Platelet-derived growth factor compositions and methods of use thereof

Compositions of platelet-derived growth factor and methods of use thereof

Field of the Invention

This invention relates to the healing of bones and connective tissues.

Background of the invention

Growth factors are proteins that bind to receptors on a cell surface, with the main result of activating cell proliferation and / or differentiation. Many growth factors are quite versatile, stimulating cell division in numerous cell types; while others are specific to a particular cell type. Examples of growth factors include platelet-derived growth factor (FCDP), insulin growth factors (FCI-I and II), beta transforming growth factor (FCT- ), epidermal growth factor (FCE) and fibroblast growth (FCF). FCDP is a heat-stable cationic protein found in a variety of cell types, including circulating platelet granules, vascular smooth muscle cells, endothelial cells, macrophage and keratinocytes and is known to stimulate protein synthesis and production in vivo. of collagen by fibroblasts. It is also known to act as an in vitro mitogen and chemotactic agent for fibroblasts, lysis muscle cells, osteoblasts and glial cells.

Recombinant human FCDP-BB (FCDP-BBhr) has been shown to stimulate wound healing bone regeneration in both animals and humans. Both in the United States and in Europe, it has been approved for human use in topical applications to accelerate the healing of foot ulcers in chronic diabetics. Recombinant FCDP-BBh has also proven effective alone or in combination with other growth factors to improve periodontal regeneration, that is, bone rebirth, dental cement or ligament around the teeth (see, for example, U.S. Patent No. 5,124,316.

US 6180606 discloses an osteogenic composition comprising a porous or semiporous matrix; demineralized bone particles; and, at least one growth factor such as PMO or beta FCT.

Summary of the Invention

We have now shown that a low dose of FCDPhr (~ 0.1 to 1.0 mg / mL) promotes bone, periodontium, ligament and cartilage repair. A low amount of FCDPhr. A low amount of FCDPhr can be adsorbed by FCT- , which can be implemented at the repair site, so that the FCDPhr is released in vivo. The addition of FCDPhr to FCT- has been shown to improve osteoblast cell binding and proliferation compared to untreated FCT- .

The present invention relates to an implant material comprising a porous demineralized bone which has a liquid incorporated therein comprising a platelet-derived growth factor (FCDP) in a concentration in the range of approximately 0.1 mg / mL at about 1.0 mg / mL, where the demineralized bone comprises particles in a range of about 100 microns to about 500 microns in size.

The invention also presents a method for promoting the growth of bone, periodontium, ligament or cartilage in a mammal, for example, a human, by administering an implant material containing platelet-derived growth factor (FCDP) in a concentration. less than 1.0 mg / ml, so that the implant material promotes the growth of bone, periodontium, ligament or cartilage. In one embodiment, the FCDP is administered in an amount less than or equal to 0.3 mg / ml. In another embodiment, the FCDP is administered in an amount in the range of about 0.1 to about 1.0 mg / ml. In several embodiments, the FCDP is administered in an amount of between about 0.2 to about 0.75 mg / ml, about 0.25 to about 0.6 mg / ml, and about 0.25 to about 0.5 mg / ml In one embodiment, the FCDP is administered in an amount of about 0.1 mg / ml, 0.3 mg / ml or 1.0 mg / ml, preferably 0.3 mg / mL. In another embodiment, the FCDP is partially or substantially purified. In yet another embodiment, the FCDP is isolated or purified from other contaminants. In a further embodiment, the FCDP is released from the implant material after administration at an average rate of 0.3 mg / day. In another embodiment, the FCDP is released from the implant material after administration at an average rate of 300 μg / day. In yet another embodiment, the FCDP is released from the implant material at an average rate of less than 100 μg / day, less than 50 μg / day, less than 10 μg / day or less than 1 μg / day. Preferably, the FCDP is administered for a few days, for example, 1, 2, 5, 10, 15, 20 or 25 days, up to 28 days or more.

The disclosure also provides a method to promote the growth of bone, periodontium, ligament or cartilage in a mammal, for example, a human, by administering an implant material containing an amount of platelet-derived growth factor (FCDP) less than approximately 1.0 mg / ml and a pharmaceutically acceptable carrier such that the implant material promotes bone growth, periodontium, ligament or cartilage and allowing bone, periodontium, ligament or cartilage to grow. Preferably, the FCDP is equal to or less than 0.3 mg / ml. In one embodiment, the FCDP is administered in a range of about 0.1 to 1.0 mg / ml. In other embodiments, the amount of FCDP is approximately 0.1 mg / ml, 0.3 mg / ml or 1.0 mg / ml, preferably 0.3 mg / mL. In another embodiment, the FCDP is partially or substantially purified. In yet another embodiment, the FCDP is isolated or purified from other contaminants. Before administering the implant material to the mammal, the method may additionally include the stage of producing a surgical skin flap to expose the bone, periodontium, ligament or cartilage and, after the administration stage, replace the flap. In yet another embodiment, after producing the surgical flap, but before administering the implant material to the bone, periodontium, ligament or cartilage, the method may additionally include the bone or periodontium cleaning step to remove organic matter from the bone or periodontium. . In yet another embodiment, the method promotes the growth of damaged or diseased bone, periodontium, ligament or cartilage. In yet another embodiment, the method promotes bone growth in locations where new bone formation is required as a result of surgical interventions, such as, for example, tooth extraction, flange augmentation, cosmetic grafting and sinus elevation.

An implant material is also provided to promote the growth of bone, periodontium, ligament

or cartilage in a mammal, for example, a human. The implant material includes a pharmaceutically acceptable carrier (for example, a biocompatible binder, a bone substitute agent, a liquid or a gel) and a platelet-derived growth factor (FCDP), which is present in a concentration lower than approximately 1.0 mg / ml Preferably, the FCDP is present in the implant material at a concentration equal to or less than 0.3 mg / ml. In one embodiment, the FCDP is administered in a range of about 0.1 to 1.0 mg / ml. In other embodiments, the amount of FCDP is approximately 0.1 mg / ml, 0.3 mg / ml or 1.0 mg / ml, preferably 0.3 mg / mL. In one embodiment, the pharmaceutically acceptable carrier of the implant material includes a framework or matrix consisting of a biocompatible binder (e.g., carboxymethyl cellulose) or a bone substitute agent (-TCP) that is capable of absorbing a solution that includes FCDP (by example, a solution containing FCDP in a concentration in the range of about 0.1 mg / mL to about 1.0 mg / mL). In another embodiment, the pharmaceutically acceptable carrier is capable of absorbing an amount of the FCDP that is equal to at least about 25% of its own weight. In other embodiments, the pharmaceutically acceptable carrier is capable of absorbing an amount of the FCDP solution that is equal to or at least about 50%, 75%, 100%, 200%, 250% or 300% or its own weight. In one embodiment, the FCDP is absorbed by the pharmaceutically acceptable carrier of the implant material by impregnating the pharmaceutically acceptable carrier in a solution containing FCDP. Preferably, the FCDP is present in a solution in a concentration of less than about 1.0 mg / mL. In another embodiment, the FCDP is present in the solution at a concentration equal to or less than about 0.3 mg / ml. In another embodiment, the FCDP is present in the solution in a concentration in the range of about 0.1 to 1.0 mg / ml. In yet another embodiment, the FCDP is present in the solution in an amount of about 0.1 mg / ml, 0.3 mg / ml or 1.0 mg / ml, preferably 0.3 mg / mL. In another embodiment, the FCDP is partially or substantially purified. In yet another embodiment, the FCDP is isolated or purified from other contaminants.

The disclosure also provides a method for preparing an implant material to promote the growth of bone, periodontium, ligament or cartilage in a mammal, for example, a human. The method includes the step of combining purified or partially purified platelet-derived growth factor (FCDP) in an amount less than about 1.0 mg / mL with a pharmaceutically acceptable carrier substance. Preferably, the FCDP is combined with a pharmaceutically acceptable carrier substance in a concentration equal to or less than about 0.3 mg / ml. In one embodiment, the FCDP is combined with a pharmaceutically acceptable carrier substance in an amount in the range of about 0.1 to 1.0 mg / ml. In other embodiments, FCDP is mixed in an amount of 0.1 mg / ml to 0.3 mg / ml, or 1.0 mg / ml. In another embodiment, FCDP is mixed in the amount 0.3 mg / ml. In yet another embodiment, the FCDP the pharmaceutically acceptable carrier absorbs the FCDP to produce the implant material.

The disclosure also provides a vial having platelet-derived growth factor (FCDP) in a concentration in the range of about 0.1 mg / mL to about 1.0 mg / mL in a pharmaceutically acceptable liquid. In one embodiment of this aspect of the invention, the liquid is sterile sodium acetate buffer. In another embodiment, the vial contains FCDP at a concentration of approximately 0.3 mg / mL. In yet another embodiment, the FCDP is FCDP-BB. In yet another embodiment, the FCDP is stable in the sodium acetate buffer for at least about 12 months, preferably at least about 18 months, more preferably at least about 24 months, and more preferably at least about 36 months when stored at a temperature in the range of about 2 ° C to 80 ° C.

The disclosure also provides an implant material that includes a porous calcium phosphate having adsorbed therein a liquid containing platelet-derived growth factor (FCDP) in a concentration in the range of about 0.1 mg / mL to about 1, 0 mg / mL The concentration of FCDP can be approximately 0.3 mg / mL, calcium phosphate can be selected from tricalcium phosphate, hydroxyapatite, low crystalline hydroxyapatite, amorphous calcium phosphate, calcium metaphosphate, dicalcium phosphate dihydrate, heptacalcium phosphate, calcium pyrophosphate dihydrate, pyrophosphate calcium and octacalcium phosphate and the FCDP can be provided in a sterile liquid, for example, sodium acetate buffer.

A method is also provided for preparing an implant material by saturating a calcium phosphate material in a sterile liquid that includes platelet-derived growth factor (FCDP) in a concentration in the range of about 0.1 mg / mL to about 1, 0 mg / mL The concentration of FCDP can be approximately 0.3 mg / mL, calcium phosphate can be selected from tricalcium phosphate, hydroxyapatite, low crystalline hydroxyapatite, amorphous calcium phosphate, calcium metaphosphate, dicalcium phosphate dihydrate, heptacalcium phosphate, calcium pyrophosphate dihydrate, pyrophosphate calcium and octacalcium phosphate.

In one embodiment of all aspects of the invention, FCDP includes homo- and heterodimers of FCDP, for example, FCDP-AA, FCDP-BB, FCDP-AB, FCDP-CC and FCDP-DD and combinations and derivatives thereof.

In an embodiment of all aspects of the invention, the pharmaceutically acceptable carrier substance of the implant material is or additionally includes one or more of the following: a biocompatible binder (for example, a natural or synthetic polymer), a bone substitute agent , a liquid and a gel. In a preferred embodiment, the implant material includes FCDP present in a pharmaceutically acceptable liquid carrier that is absorbed by a pharmaceutically acceptable solid carrier.

The implant material of the present disclosure can be prepared by combining isolated, partially purified, substantially purified or purified FCDP in an amount in the range of 0.1 to 1.0 mg / ml, more preferably 0.1 mg / ml, 0, 3 mg / ml or 1.0 mg / ml, more preferably 0.3 mg / ml, or even less than 0.1 mg / ml, with a pharmaceutically acceptable carrier substance, for example, a biocompatible binder, such as a polymer natural or synthetic (for example, collagen, polyglycolic acid and polylactic acid), a bone substitute agent (for example, calcium phosphate (for example, tricalcium phosphate or hydroxyapatite), calcium sulfate or demineralized bone (for example, demineralized lyophilized cortical or spongy bone) or gel or liquid available in the market (that is, a gel or viscous or inert liquid).

In various embodiments, the carrier substance of the implant material is, or additionally includes, one

or more biocompatible binders. A biocompatible binder is an agent that produces or promotes cohesion between the combined substances. Non-limiting examples of suitable biocompatible binders include polymers selected from polysaccharides, nucleic acids, carbohydrates, proteins, polypeptides, poly (ahydroxy acids), poly (lactones), poly (amino acids), poi (anhydrides), poly (orthoesters), poly ( anhydride-co-imides), poly (orthocarbonates), poly (a-hydroxy alkanoates), poly (dioxanones), poly (phosphoesters), polylactic acid, pol (L-lactide) (PLLA), poly (D, L-lactide ), (PDLLA), polyglycolide (PGA), poly (lactide-co-glycolide (PLGA), poly (L-lactide-co-D, L-lactide), poly (D, L-lactide-co-carbonate trimethylene), polyglycolic acid, polyhydroxybutyrate (PHB), poly ( £ -caprolactone), poly (5-valerolactone), poly (y-butyrolactone), poly (caprolactone), polyacrylic acid, polycarboxylic acid, poly (allylamine hydrochloride), poly (diallyldimethylammonium chloride), poly (ethyleneimine), polypropylene fumarate , polyvinyl alcohol, polyvinylpyrrolidione, polyethylene, polymethylmethacrylate, carbon fibers, poly (ethylene glycol), poly (ethylene oxide), polyI (vinyl alcohol), poly (vinyl pyrrolidone), poly (ethyloxazoline), poly (ethylene oxide) -copol (propylene oxide) block copolymers, poly (ethylene teraphthalate) polyamide, and copolymers and mixtures thereof. Additional binders include alginic acid, gum arabic, guar gum, xanthan gum, gelatin, chitin, chitosan, chitosan acetate, chitosan lactate, chondroitin sulfate, NO-carboxymethyl chitosan, a dextran (e.g., a-cyclodextrin, -cyclodextrin, y-cyclodextrin, or sodium dextran sulfate), fibrin glue, glycerol, hyaluronic acid, sodium hyaluronate, a cellulose (e.g., methylcellulose, carboxymethylcellulose, hydroxypropylmethylcellulose, or hydroxyethylcellulose), a glucosamine, a proteoglycan, a starch (for example, hydroxyethyl starch or soluble starch), lactic acid, a pluronic, sodium glycerophosphate, collagen, glycogen, a keratin, silk and derivatives and mixtures thereof. A water soluble binder dissolves from the implant material shortly after implantation in vivo, thereby introducing macroporosity into the implant material. This macroporosity increases the osteoconductivity of the implant material, increasing access and, as a consequence, the remodeling activity of osteoclasts and osteoblasts at the implant site.

The biocompatible binder can be added to the implant material in various amounts and in a variety of phases during the preparation of the composition. Those skilled in the art will be able to determine the amount of binder and the method of inclusion required for a given application.

In one embodiment, the carrier substance is, or includes, a liquid selected from water, a buffer, and a cell culture medium. The liquid can be used in any pH range, but will be used more frequently in the range of pH 5.0 to pH 8.0. In one embodiment, the pH will be compatible with the prolonged stability and efficacy of the FCDP present in the implant material, or with the prolonged stability and efficacy of another biologically active agent. In most embodiments, the pH of the liquid will be in the range of pH 55 to pH 7.4. Suitable buffers include, but are not limited to, carbonates, phosphates (eg, phosphate buffered saline), and organic buffers such as Tris, HEPES and MOPS. More frequently, the buffer will be selected for its biocompatibility with host tissues and its compatibility with the biologically active agent. For most applications in which nucleic acids, peptides or antibiotics are included in the implant material, a simple phosphate buffered buffer will suffice.

In another embodiment of all aspects of the invention, the carrier substance of the implant material is, or additionally includes, one or more bone substitute agents. A bone substitute agent is one that can be used to replace a bone permanently or temporarily. After implantation, the body can retain the bone substitute agent or the body can absorb it and replace it with bone. Exemplary bone substitute agents include, for example, calcium phosphate (e.g., tricalcium phosphate (e.g., -TCP), hydroxyapatite, low crystalline hydroxyapatite, amorphous calcium phosphate, calcium metaphosphate, dicalcium phosphate dihydrate, heptacalcium phosphate, calcium pyrophosphate, calcium pyrophosphate and octacalcium phosphate), calcium sulfate, demineralized bone (for example, demineralized lyophilized cortical or spongy bone)). In one embodiment, the transportor and bio-absorbable substance. In another embodiment, the bone substitute agent is provided as a matrix of micron or submicron-sized particles, for example, nano-sized particles. The particles may be in the range of about 100 µm to about 500 µm in size, more preferably in the range of about 200 µm to about 3000 µm, and more preferably in the range of about 250 µm to about 2000 µm, or the particles they may be in the range of about 1 mm to about 10,000 nm, preferably less than about 500 nm, and more preferably less than about 250 nm. In another embodiment, the bone substitute agent has a porous composition. The porosity of the composition is a desirable feature since it facilitates migration and cell infiltration into the composition so that cells can secrete extracellular bone matrix. It also provides access to vascularization. Porosity also provides a high surface area for better reabsorption and release of active substances, as well as greater cell-matrix interaction. Preferably, the composition has a porosity greater than 40%, more preferably greater than 65% and more preferably greater than 90%. The composition can be provided in a form suitable for implantation (for example, a sphere, a cylinder or a block) or it can be given the size and shape before use.

The bone substitute agent can also be provided as a fluid and moldable paste or putty. The bone substitute agent may be a calcium phosphate paste that hardens itself to form a hardened calcium phosphate before or after implantation in vivo. The calcium phosphate component may be any biocompatible calcium phosphate material known in the art. The calcium phosphate material can be produced by a variety of methods and using any suitable initial component. For example, the calcium phosphate material may include amorphous, apatitic calcium phosphate. The calcium phosphate material can be produced by a solid-state acid-base reaction of crystalline calcium phosphate reagents to form crystalline hydroxyapatatite solids. Other methods for making calcium phosphate materials are known in the art, and some of which are described below.

The calcium phosphate material may be low crystalline apatitic calcium phosphate (APC) or hydroxyapataite (HA). The APC material is described in the application for US patent numbers 5,650,176; 5,783,217; 6,027,742; 6,214,368; 6,287,341; 6,331,312 and 6,541,037. HA is described, for example, in US Patent Numbers Re. 33,221 and Re. 33,161. These patents show the preparation of remineralization compositions of calcium phosphate and gradually resorbable, finely crystalline and non-ceramic hydroxyapatite transport material based on the same calcium phosphate composition. A similar calcium phosphate system, which consists of tetracalcium phosphate (TTCF) and monocalcium phosphate (MCF) or its monohydrate form (MCFM), is described in US Patent Nos. 5,053,212 and 5,129,905. This calcium phosphate material is produced by acid-base solid-state reaction of crystalline calcium phosphate reagents to form crystalline hydroxyapatatite solids.

Crystalline HA materials (commonly referred to as dalite) can be prepared in such a way that they are fluid, moldable and capable of hardening in situ (see US Patent Number 5,962,028). These HA materials (commonly referred to as carbonated hydroxyapatite) can be formed by combining the reagents with a non-aqueous liquid to provide a substantially uniform mixture, shaping the mixture as appropriate, and allowing the mixture to harden in the presence of water (for example , before or after the implant). During hardening, the mixture crystallizes into a solid and essentially monolithic apatite structure.

The reagents will generally consist of a phosphate source, for example, phosphoric acid or phosphate salts, substantially free of water, an alkali earth metal, particularly calcium, source, optionally crystalline nuclei, particularly calcium phosphate or hydroxyapatite crystals, calcium carbonate and physiologically acceptable lubricant, such as any of the non-aqueous liquids described herein. The dry ingredients can be prepared previously as a mixture and subsequently combined with the non-aqueous liquid ingredients under conditions where a substantially uniform mixture occurs.

The calcium phosphate material is characterized by its biological resorbability, biocompatibility and its minimal crystallinity. Its crystalline character is substantially the same as that of natural bone. Preferably, the calcium phosphate material hardens in less than five hours, and substantially hardens in approximately one to five hours, under physiological conditions. Preferably, the material hardens substantially in about 10-30 minutes. The speed of hardening under physiological conditions may vary according to the therapeutic need by modifying a few simple parameters as described in US Patent No. 6,027,742.

In one embodiment, the resulting bioresorbable calcium phosphate material will be "calcium deficient," with a calcium to phosphate molar ratio of less than about 1.6 in comparison to the ideal stoichiometric value of about 1.67 for hydroxyapatatia.

Desirable calcium phosphates are capable of hardening in a humid medium, at or around body temperature in less than 5 hours and preferably in 10-30 minutes. Desirable materials are those that, when implanted as a 1-5 g pellet, at least 80% are reabsorbed in a year. Preferably, the material can be completely reabsorbed.

In various embodiments of all aspects of the invention, the implant material may additionally include one or more biologically active agents. Biologically active agents that can be incorporated into the implant materials of the invention include, without limitation, organic molecules, inorganic materials, proteins, peptides, nucleic acids (e.g., genes, gene fragments, gene regulatory sequences and antisense molecules) , nucleoproteins, polysaccharides, glycoproteins and lipoproteins. The classes of biologically active compounds that can be incorporated into the implant materials of the invention include, without limitation, anti-cancer agents, antibiotics, analgesics, anti-inflammatory agents, immunosuppressants, enzyme inhibitors, antihistamines, anti-convulsants, hormones, muscle relaxants. , antispasmodic substances, ophthalmic agents, prostaglandins, anti-depressants, anti-psychotic substances, trophic factors, osteoinductive proteins, growth factors and vaccines.

Anti-cancer agents include alkylating agents, platinum agents, antimetabolites, topoisomerase inhibitors, antitumor antibiotics, antimitotic agents, aromatase inhibitors, thymidylate synthase inhibitors, DNA antagonists, farnesyltransferase inhibitors, pump inhibitors, acetyltransferase inhibitors of histone, metalloproteinase inhibitors, ribonucleoside reductase inhibitors, TNF alpha agonists / antagonists, Endothelium A receptor antagonists, retinoic acid receptor agonists, immunomodulators, hormonal and antihormonal agents, photodynamic agents and tyrosine kinase inhibitors.

Any of the biologically active agents listed in Table 1 may be used.

Table 1

<dl><dt>Alkylating agents </dt><dd>cyclophosphamide Busulfan ifosfamide melphalan hexamethylmelamine thiotepa chlorambucil dacarbazine carmustine Lomustine Procarbazine Altretamine Estramustine Phosphate Mechlorethamine Streptozocin Temozolamide Semustine </dd></dl>

&lt;p /&gt;

<dl><dt>Platinum Agents </dt><dd>cisplatin oxaliplatin spiroplatin carboxiftalate platinum tetraplatin ormiplatin iproplatin carboplatin ZD-0473 (AnorMED) lobaplatin (Aeterna) satraplatin (Johnson Matthey) BBR-3464 (Hoffmann-La Roche) SM-11355 (Sumitomo) AP-5289 (Access) </dd></dl>

&lt;p /&gt;

<dl><dt>Antimetabolites </dt><dd>azacitidine gemcitabine capecitabine 5-flurorouracil Floxuridine 2-chlorodeoxyadenosine 6-mercaptopurine 6-thioguanine cytarabine 2-fluorodeoxy citidine methotrexate idatrexate tomudex trimetrexate deoxycoformycin fludarabine pentostatin raltitrexed hydroxyurea deciatin (SuperGen) clofarabine (Bioenvision) irofulven (MGI Pharma) DMDC (Hoffmann-La Roche) ethinylcitidine (Taiho) </dd></dl>

&lt;p /&gt;

<dl><dt>Topoisomerase inhibitors </dt><dd>amsacrine epirobicin terniposide ethoposide or irinotecan mitoxantrone (CPT-11) 4-ethyl-10-hydroxy-campotecina Topotecan dexrazoxanet (TopoTarget) pixantrone (Novuspharma) rebecamycin analogue (Exelixis) BBR-3576 (Novuspharma) Rubitecan (SuperGen) exatecan mesylate (Daiichi) quinamed (ChemGenex) gimatcan (Sigma-Tau) diflomotecano (Sigma-Tau) TAS-103 (Taiho) elsamitrucine (Spectrum) J-107088 (Merck &amp; Co) BNP-1350 (BioNumerik) CKD -602 (Chong Kun Dang) KW-2170 (Kyowa Hakko) </dd></dl>

&lt;p /&gt;

<dl><dt>Tumor Antibiotics </dt><dd>dactinomycin (actinomycin D) doxorubicin (adriamycin) doxirubicin valrubicin daunorubicin (daunomycin) epirubicin terarubicin idarubicin rubidazone plicamycin porphyromycin cyanomorpholinodoxorubicin mitoxantrone (novantrone) amonafida azonafida antrapirazol oxantrazole losoxantrone bleomycin sulfate (blenoxane) bleomycinic acid bleomycin A bleomycin B mitomycin C MEN-10755 (Menarini) GPX-100 (Gem Pharmaceuticals) </dd></dl>

&lt;p /&gt;

<dl><dt>Antifungal agents </dt><dd>paclitaxel docetaxel colchicine vinblastine vincristine vinorelbine Vindestine dolastatin 10 (NCI) rhizoxin (Fujisawa) mivobulin (Warner-Lambert) cemadotine (BASF) RPR 109881A (Aventis) TXD 258 (Aventis) epothilone B (Novartis) T 900607 (Tik Tikular) ) Cryptophytic 52 (Eli Lilly) Vinflunine (Fabre) Auristatin PE (Teikoku Hormone) BMS 247550 (BMS) BMS 184476 (BMS) BMS 188797 (BMS) Taxoprexin (Protarga) SB 408075 (GlaxoSmithKline) E7010 (Abbott) PG-TXL (Cell Therapeutics) IDN 5109 (Bayer) A 105972 (Abbott) A 204197 (Abbott) LU 223651 (BASF) D 24851 (ASTAMedica) ER-86526 (Eisai) combretastatin A4 (BMS) isohomohalicondrine-B (PharmaMar) ZD 6126 (AstraZeneca) PEG-paclitaxel (Enzon ) AZ10992 (Asahi) IDN-5109 (Indena) AVLB (Prescient NeuroPharma) azaepotilone B (BMS) BNP-7787 (BioNumerik) prodrug CA-4 (OXiGENE) dolastatin-10 (NIH) CA-4 (OXiGENE) </dd></dl>

&lt;p /&gt;

<dl><dt>Aromatase inhibitors </dt><dd>aminoglutehimide letrozole anastrazole formestane atamestane exemestane (BioMedicines) YM-511 (Yamanouchi) </dd></dl>

&lt;p /&gt;

<dl><dt>Thymidylate synthase inhibitors </dt><dd>pemetrexed (Eli Lilly) ZD-9331 (BTG) nolatrexed (Eximias) CoFactor ™ (BioKeys) </dd></dl>

&lt;p /&gt;

<dl><dt>DNA antagonists </dt><dd>trabectedin (PharmaMar) glufosfamide (Baxter International) albumin + 32 P (Isotope Solutions) timectacin (NewBiotics) edotreotide (Novartis) mafosfamide (Baxter International) apaziquone (Spectrum Pharmaceuticals) O6 guanine benzyl (Paligent) </dd></dl>

&lt;p /&gt;

<dl><dt>Farnesitransferase inhibitors </dt><dd>arglabina (NuOncology Labs) ionafarnib (Schering-Plow) BAY-43-9006 (Bayer) tripifarnib (Johnson &amp; Johnson) alcohol perilil (DOR BioPharma) </dd></dl>

&lt;p /&gt;

<dl><dt>Pump inhibitors </dt><dd>CBT-1 (CBA Pharma) Tariquidar (Xenova) MS-209 (Schering AG) Zosuquidar thihydrochloride (Eli Lilly) biricodar dicitrate (Vertex) </dd></dl>

&lt;p /&gt;

<dl><dt>Histone Acetyltransferase Inhibitors </dt><dd>Tacedinaline (Pfizer) SAHA (Aton Pharma) MS-275 (Schering AG) Pivaloyloxymethyl butyrate (Titan) depsypeptide (Fujisawa) </dd></dl>

&lt;p /&gt;

<dl><dt>Metalloproteinase inhibitors </dt><dd>neovastat (Aeterna Laboratories) marimastat (British Biotech) CMT-3 (CollaGenex) BMS-275291 (Celltech) </dd></dl>

&lt;p /&gt;

<dl><dt>Ribonucleoside reductase inhibitors </dt><dd>Gallium Malonate (Titan) Triapine (Vion) tezacitabine (Aventis) didox (Molecules for Health) </dd></dl>

&lt;p /&gt;

<dl><dt>TNF alpha agonists / antagonists </dt><dd>virulicin (Lorus Therapeutics) CDC-394 (Celgene) infliximab (Centocor, Inc.) adalimumab (Abbott Laboratories) revimid (Celgen) entanercept (Immunex Corp.) </dd></dl>

&lt;p /&gt;

<dl><dt>Endothelium A receptor antagonists </dt><dd>atrasentan (Abbott) ZD-4054 ​​(AstraZeneca) YM-598 (Yamanouchi) </dd></dl>

&lt;p /&gt;

<dl><dt>Retinoic acid receptor agonists </dt><dd>Pheretinide (Johnson &amp; Johnson) LGD-1550 (Ligand) alitretinoin (Ligand) </dd></dl>

&lt;p /&gt;

<dl><dt>Immunomodulators</dt><dd> Oncofago interferon (Antigenics) GMK (Progenics) adenocarcinomas vaccine (Biomira) CTP-37 (AVI BioPharma) IRX-2 (Immuno-Rx) PEP-005 (Peplin Biotech) syncrovax vaccines (CTL Immuno) melanoma vaccine (CTL Immuno) vaccine p21 RAS (GemVax) dexosome therapy (Anosys) Pentrix (Australian Cancer Technology) ISF-154 (Tragen) cancer vaccine (Intercell) Norelin (Biostar) BLP-25 (Biomira) MGV (Progenics) -aletine (Dovetail) CLL therapy (VAsogen) </dd></dl>

&lt;p /&gt;

<dl><dt>Hormonal and antihormonal agents </dt><dd>estrogens conjugated estrogens ethinyl estradiol chlortrianisene idenestrol hydroxyprogesterone caproate medroxyprogesterone testosterone testosterone propionate fluoxymesterone methyltestoserone diethylstilbestrol megestol tamoxifen toremophin dexamethasone prednisone methylprednisone prednisolone aminoglutethimide leuprolide goserelin leuporelin bicalutamide flutamide octreotide nilutamide mitotane P-04 (Novagen) 2-methoxystradiol (EntreMed) Arzoxifene (Eli Lilly) </dd></dl>

<dl><dt>Photodynamic agents </dt><dd>talaporfina (Light Sciences) Theralux (Theratechnologies) gadolinium motexafina (Pharmacyclics) Pd-bacteriofeoforbide (Yeda) lutetium texaphyrin (Pharamacyclics) hypercirin </dd></dl>

&lt;p /&gt;

<dl><dt>Tyrosine Kinase Inhibitors </dt><dd>Imatinib (Novartis) Leflunomide (Sugen / Pharmacia) ZD1839 (AstraZeneca) Erlotinib (Oncogene Science) Canertinib (Pfizer) Squalamine (Genaera) SU5416 (Pharmacia) SU6668 (Pharmacia) ZD4190 (AstraZeneca) ZD64ca (AstraZertis Novart (PK6) ) GW2016 (GlaxoSmithKline) EKB-509 (Wyeth) EKB-569 (Wyeth) kahalido F (PharmaMar) CEP-701 (Cephalon) CEP-751 (Cephalon) MLN518 (Millenium) PKC412 (Novartis) phenoxy diol () trastuzumab (Genetech) C225 (ImClone) Ru-Mab (Genentech) MDX-H210 (Medarex) 2C4 (Genentech) MDX-447 (Medarex) ABX-EGF (Abgenix) IMC-1C11 (ImClone) </dd></dl>

Antibiotics include aminoglycosides (for example, gentamicin, tobramycin, netilmicin, streptomycin, amicacin, neomycin), bacitracin, carbapenemas (for example imipenem / cilastatin), cephalosporins, colistin, methenamine, monobactams (for example, aztreonam (penic for example) , penicillin G, penicicline V, methicillin, natillin, oxacillin, cloxacillin, dicloxacillin, ampicillin, amoxicillin, carbenicillin, ticarcillin, piperacillin, mezlocillin, azlocillin), polymyxin B, quinolones and vancomycin; and bacteriostatic agents such as chloramphenicol, clindanyan, macrolides (for example, erythromycin, azithromycin, clarithromycin), lincomycin, nitrofurantoin, sulfonamides, tetracyclines (for example, tetracycline, doxycycline, minocycline, demeclocylin) and trimethoprim. Metronidazole, fluoroquinolones and ritampin are also included.

Enzyme inhibitors are substances that inhibit an enzymatic reaction. Examples of enzyme inhibitors include edrophonium chloride, N-methylphysostigmine, neostigmine bromide, physostigmine sulfate, tacrine, tacrine, 1-hydroxy maleate, iodotubercidine, p-bromotetramisole, 10- (alpha-diethylaminopropionyl) -phenotiacin hydrochloride, chloride calmidazolium, hemicolinium-3,3,5-dinitrocatecol, diacylglycerol kinase I inhibitor, diacylglycerol kinase II inhibitor, 3-phenylpropargilamine, N6-monomethyl-L-arginine acetate, carbidopa, 3-hydroxybenylhydrazine, hydralazine, clorgiline, deprenyl, hydroxylamine, iproniazide phosphate, 6-MeO-tetrahydro-9H-pyrido-indole, nialamide, pargiline, quinacrine, semicarbacid, tranylcypromine, N, N-diethylaminoethyl-2,2-diphenylvalerate hydrochloride, 3-isoiso -1-methylxantane, papaerine, indomethacin, 2-cyclooctyl-2-hydroxyethylamine hydrochloride, 2.32-dichloro-a-methylbenzylamine (DCMB), 8,9-dichloro-2,3,4,5-tetrahydro-1H-2-benzacepin hydrochloride, p-aminoglutethimide, p-aminoglutethimide tartrate, 3-iodotyrosine, alpha-methyl tyrosine, acetazolamide, dichlorphenamide, 6-hydroxy-2-benzothiazolosulfonamide and allopurinol.

Antihistamines include pyrilamine, chlorpheniramine and tetrahydrozoline, among others.

Anti-inflammatory agents include corticosteroids, non-steroidal anti-inflammatory drugs (for example, aspirin, phenylbutazone, indomethacin, sulindac, tolmetine, ibuprofen, piroxicam and phenamates), acetaminophen, phenacetin, gold salts, chloroquine, D-Penicillamine, methotrexate, methotrexate, methotrexate , probenecid and sulfinpyrazone.

Muscle relaxants include mefenesin, methocarbomal, cyclobenzaprine hydrochloride, trihexylphenidyl hydrochloride, levodopa / carbidopa and biperidene.

Anti-spasmodics include atropine, scopolamine, oxyphenonium and papaverine.

Painkillers include aspirin, phenylbutazone, indomethacin, sulindac, tolletic, ibuprofen, piroxicam, phenamates, acetaminophen, phenacetin, morphine sulfate, codeine sulfate, meperidine, nalorphine, opioids (for example, codeine sulfate, fentarate citrate, bonatarate hydrocodone, loperamide, morphine sulfate, noscapine, norcodeine, normorphine, thebaine, nor-binaltorphimine, buprenorphine, clornaltrexamine, funaltrexamione, nalfufine, narlofine, naloxin, naloxonazine, Naltrexone and Naltrindole, Procaine, Lidocaine, Tetracaine and Dibucaine.

Ophthalmological agents include sodium fluorescein, rose bengal, methacholine, adrenaline, cocaine, atropine, alpha-chymotrypsin, hyaluronidase, betaxolol, pilocarpine, timolol, timolol salts and combinations thereof.

Prostaglandins are recognized in the art and are a class of long chain hydroxy fatty acids that occur naturally chemically related.

Antidepressants are substances capable of preventing or releasing depression. Examples of antidepressants include imipramine, amitriptyline, nortriptyline, protriptyline, desipramine, amoxapine, doxepine, maprotiline, tranylcypromine, phenelcin and isocarboxazide.

Growth factors are factors whose continued presence improves the viability or longevity of a cell. Trophic factors include, without limitation, neutrophil activating protein, monocyte chemoattractant protein, macrophage inflammatory protein, platelet factor, basic platelet protein and melanoma growth stimulating activity; epidermal growth factor, transforming growth factor (alpha), fibroblast growth factor, platelet-derived endothelial cell growth factor, insulin growth factor (FCI, for example, FCI-I or FCI-II), neurotrophic factor glial derived, ciliary neurotrophic factor, nerve growth factor, bone / cartilage growth inducing factor (alpha and beta), bone morphogenetic proteins (PMOs), interleukins (interleukin inhibitors or interleukin receptors, including from interleukin 1 to interleukin 10), interferons (e.g., alpha interferon, beta and gamma), hematopoietic factors, including erythropoietin, granulocyte colony stimulating factor, colony stimulating factor macrophages and granulocyte-macrophage colony stimulating factor; tumor necrosis factors, transforming growth factors (beta), including beta-1, beta-2, beta-3, transforming growth factors (alpha), inhibin and activin; and bone morphogenetic proteins such as OP-1, PMO-2 and PMO-7.

The hormones include estrogens (e.g., estradiol, estrone, estriol, dieteilestbestrol, kinestro, chlorotrianisen, ethinyl estradiol, mestranol), anti-estrogens (e.g., clomiphene, tamoxifen), progestins (e.g., medroxyprogesterone, noretindronone, hydroxyprogesterone, hydroxyprogesterone, hydroxyprogesterone, hydroxyprogesterone, hydroxyprogesterone, hydroxyprogesterone, hydroxyprogesterone, hydroxyprogesterone, hydroxyprogesterone, hydroxyprogesterone, hydroxyprogesterone, hydroxyprogesterone, hydroxyprogesterone, hydroxyprogesterone, hydroxyprogesterone, hydroxyprogesterone, hydroxyprogesterone, hydroxyprogesterone, hydroxyprogesterone, hydroxyprogesterone, hydroxyprogesterone, hydroxyprogesterone, hydroxyprogesone , antiprogestin (mifepristone), androgens (for example, testosterone cypionate, fluoxymesterone, danazol, testolactone), anti-androgens (for example, cyproterone acetate, flutamide), Thyroid hormones (for example, triiodothyronine, thyroxine, propylthiouracil, methimazole and iodixoda), and pituitary hormones (for example, corticotropin, sumutotropin, oxytocin and vasopressin). Hormones are commonly used in hormone replacement therapy and / or for contraceptive purposes. Steroid hormones, such as prednisone, are also used as immunosuppressants and anti-inflammatories.

The biologically active agent is desirably selected from the family of proteins known as transforming growth factors -beta (FCT-) protein superfamily, which includes activins, inhibins and bone morphogenetic proteins (PMOs). In one embodiment, the active agent includes at least one protein selected from the subclass of proteins generally known as PMO, which have been described as having osteogenic activity, and other growth and differentiation type activities. These PMOs include PMO proteins PMO-2, PMO-3, PMO-4, PMO-5, PMO-6 and PMO-7, described for example in US Patent No. 5,108,922; 5,013,649; 5,116,738; 5,106,748; 5,187,076; and 5,141,905; PMO-8, is described in PCT publication WO91 / 18098, and PMO-9, described in PCT publication WO93 / 00432, PMO-10, described in PCT application WO94 / 26893; PMO-11, described in PCT application WO94 / 26892, or PMO-12 or PMO-13, described in PCT application WO 95/16035; PMO-14; PMO-15, described in US Patent No. 5,635,372; or PMO-16, described in U.S. Patent No. 5,965,403. Other FCT- proteins that may be useful as an active agent in the calcium phosphate compositions of the invention include Vgr-2, Jones et al., Mol. Endocrinol 6: 1961 (1992), and any of the growth and differentiation factors (CDFs), including those described in PCT applications WO94 / 15965; WO94 / 15949; WO95 / 01801; WO95 / 01802; WO94 / 21681; WO94 / 15966; WO95 / 10539; WO96 / 01845; WO96 / 02559 and others. Also useful in the invention may be BIP, described in WO94 / 01557; HP00269, described in JP Publication number: 7-250688, and MP52, described in PCT application WO93 / 16099. A subset of the PMO that is currently preferred for use in the invention includes PMO-2, PMO-4, PMO-5, PMO-6, PMO-7, PMO-10, PMO-12, PMO-13, PMO- 14, and MP52. The active agent is more preferably PMO-2, the sequence of which is described in United States Patent No. 5,013,649. Other osteogenic agents known in the art may also be used, such as teriparatide (Forteo ™), Chrysalin®, prostaglandin E2, LIM protein, osteogenin, or demineralized bone matrix (MOD), among others.

The biologically active agent can be chemically synthesized, recombinantly produced, or purified from a source in which the biologically active agent is found naturally. The active agent, if an FCT-comotal such as a PMO, or other dimeric protein, can be homodimeric, or it can be heterodimeric with other PMOs (for example, a heterodimer composed of a monomer of each of PMO-2 and PMO- 6) or with other members of the FCT-super superfamily, such as activins, inhibins and FCT-1 (for example, a heterodimer consisting of a monomer of each of a PMO and a related member of the FCT superfamily

). Examples of such heterodimeric proteins are described for example in published PCT patent application WO 93/09229.

Additional biologically active agents include Hedgehog, Frazzled, Chordin, Noggin, Cerberus and folistatin proteins. These protein families are generally described in Sasai et al., Cell 79: 779-790 (1994) (Chordin); PCT patent publication WO94 / 05800 (Noggin); and Fukui et al, Devel .. Biol. 159: 131 (1993) (Folistatina). Hedgehog proteins are described in WO96 / 16668; WO96 / 17,924, and WO95 / 18856. The Frazzled family of proteins is a recently discovered family of proteins with high homology with the extracellular binding domain of the receptor protein family known as Frizzled. The Frizzled family of genes and proteins is described in Wang et al., J. Biol. Chem. 271: 4468-4476 (1996). The active agent may also include other soluble receptors, such as the truncated soluble receptors described in PCT patent publication WO95 / 07982. From the teaching of WO95 / 07982, one skilled in the art will recognize that truncated soluble receptors can be prepared for numerous other receptor proteins.

The amount of biologically active protein, for example, an osteogenic protein, which is effective to stimulate a desired activity, for example, increased osteogenic activity of progenitor cells present or infiltrators or other cells will depend on the size and nature of the defect being treated. , as well as the conveyor being used. Generally, the amount of protein to be administered is in a range of about 0.1 to about 100 mg, preferably about 1 to about 100 mg; more preferably from about 10 to about 80 mg.

Standard protocols and regimes for the administration of the agents listed above are known in the art. Biologically active agents are introduced into the implant material in amounts that allow the administration of an appropriate dose of the agents at the implant site. In most cases, doses are determined using guidelines known to doctors and applicable to the particular agent in question. The exemplary amount of biologically active agent to be included in the implant material of the invention will probably depend on variables such as type and extent of the condition, the general state of health of the particular patient, the formulation of the active agent and the bioreabsorption of the vehicle. of administration used. Standard clinical trials can be used to optimize the dose and dose frequency for a particular biologically active agent.

In an embodiment of all aspects of the invention, the composition may also contain autologous bone marrow or autologous platelet extracts.

In another embodiment of all of the above aspects, the FCDP and / or other growth factors can be obtained from natural sources (eg, platelets), or more preferably, produced by recombinant DNA technology. When obtained from natural sources, the FCDP and / or other growth factors can be obtained from a biological fluid. A biological fluid includes any treated or untreated fluid (including a suspension) associated with living organisms, particularly blood, including whole blood, hot or cold blood, and stored or fresh blood; treated blood, such as blood diluted with at least one physiological solution, including but not limited to saline, nutrient and / or anticoagulant solutions; blood components, such as platelet concentrate (CP), platelets under apheresis, platelet-rich plasma (PRP), platelet-poor plasma (PPP), platelet-free plasma, plasma, serum, fresh frozen plasma (PCF) , components obtained from plasma, packed red blood cells (GRE), leukocyte layer (CL); blood products derived from blood or a component of blood or bone marrow derivatives; red blood cells separated from plasma and resuspended in physiological fluid; and platelets separated from plasma and resuspended in physiological fluid. The biological fluid may have been treated to remove some of the leukocytes before being processed according to the invention. As used herein, the blood product or biological fluid refers to the components described above, and similar blood products or biological fluids obtained by other means and with similar properties. In one embodiment, the FCDP is obtained from platelet rich plasma (PRP). The preparation of PRP is described, for example, in U.S. Patents Nos. 6,649,072, 6,641,552, 6,613,566, 6,592,507, 6,558,307, 6,398,972 and 5,599,558.

In one embodiment of all aspects of the invention, the implant material administers FCDP to the implant site for a period of time greater than at least 1 day. In several embodiments, the implant material administers FCDP to the implant site for at least 7, 14, 21 or 28 days. Preferably, the implant material administers FCDP to the implant site for a time between approximately 1 day and 7, 14, 21 or 28 days. In another embodiment, the implant material administers FCDP to the implant site for a time greater than about 1 day, but less than about 14 days.

By "resorbable" is meant the ability of the implant material to be absorbed or remodeled again in vivo. The reabsorption process involves the degradation and elimination of the original implant material through the action of body fluids, enzymes or cells. The host can use the resorbed materials in the formation of new tissue, or the host can use them in another way, or they can be excreted.

By "differentiation factor" is meant a polypeptide, including a chain of at least 6 amino acids, that stimulates the differentiation of one or more target cells into cells with the potential to form cartilage or bone.

By "particles of nanometer size" is meant a particle of submicron size, generally defined as a particle below 1000 nanometers. A particle of nanometer size is a solid particle material that is an intermediate state between molecular substances and micron. A nanometer is defined as one billionth of a meter (1 nanometer = 109 m). The nanometer material is known as powder, fiber, film or block has a nanoscale size.

By "periodontium" is meant the tissues that surround and hold the teeth. The periodontium holds, protects and provides nutrients to the teeth. The periodontium consists of bone, cement, alveolar process of the jaws and jaw, periodontal ligament and gum. Cement is a thin, calcified layer of tissue that completely covers the dentin of the tooth root. Cement is formed during root development and during the life of the tooth and functions as an area of ​​union of periodontal ligament fibers. The alveolar process is the bony part of the jaw and jaw where the teeth are embedded and where the roots of the teeth are attached. The socket is the cavity where the alveolar process in which the root of the tooth is maintained by the periodontal ligament. The bone that divides one socket from another is called the interdental septum. When teeth with multiple roots are present, the bone is called interradicular septum. The alveolar process includes the cortical plate, alveolar crest, trabecular bone and the appropriate alveolar bone.

By "promoting growth" is meant the healing of bone, periodontium, ligament or cartilage, and regeneration of such tissues and structures. Preferably, the bone, periodontium, ligament or cartilage is damaged or injured and requires regeneration or healing.

"Promoting the growth of the periodontium" means the regeneration or healing of the supporting tissues of a tooth including alveolar bone, cement and interposed periodontal ligament, which have been damaged by disease or trauma.

By "purified" is meant a growth or differentiation factor, for example, FCDP, which, before mixing with a carrier substance, is 95% or greater by weight, that is, the factor is substantially free of other proteins, lipids and Carbohydrates with what is naturally associated. The term "substantially purified" refers to a lower factor purity, which has, for example, only 5% -95% by weight of the factor, preferably 65-95%. A purified protein preparation will generally produce a single major strip on a polyacrylamide gel. More preferably, the purified factor used in implant materials of the invention is pure when judged by the amino terminal amino acid sequence analysis. The terms "partially purified" refer to FCDP that is provided in the context of PRP, PPP, PCF, or any other blood product that requires collection and separation to occur, for example, by centrifugation.

By way of example, a solution having ~ 1.0 mg / mL of FCDP, when ~ 50% is pure, constitutes ~ 2.0 mg / mL of total protein.

The implant materials of this invention aid in the regeneration of the periodontium, at least in part, by promoting the growth of connective tissue, bone and cement. Implant materials can be prepared to directly promote the growth and differentiation of cells that produce connective tissue, bone and cement. Alternatively, implant materials can be prepared to act indirectly, for example, by attracting cells that are necessary to promote the growth of connective tissue, bone and cement. Regeneration using a composition of this invention is a more effective treatment of periodontal diseases or bone wounds than that achieved using systemic antibiotics or only surgical debridement.

The FCDP, polypeptide growth factors, and differentiation factors can be obtained from human tissues or cells, for example, platelets, by solid phase peptide synthesis, or by recombinant DNA technology. Thus, with the terms "polypeptide growth factor" or "differentiation factor", recombinant or synthesized materials derived from tissue or cells are understood. If the factor is a dimer, for example, FCDP, the recombinant factor can be a recombinant heterodimer, made by inserting into DNA prokaryotic or eukaryotic cells that encode both subunits of the factor, and then allowing the cells to process the subunits transferred to form a heterodimer (for example, FCDP-AB). Alternatively, DNA encoding only one of the subunits (for example, FCDP chain B or chain A) can be inserted into cells, which are then cultured to produce the homodimeric factor (for example, FCDP-BB or FCDP-AA homodimers). FCDP for use in the methods of the invention includes homo and FCDP heterodimers, for example, FCDP-AA, FCDP-BB, FCDP-AB, FCDP-CC and FCDP-DD, and combination and derivatives thereof.

The concentration of FCDP and other growth factors of the invention can be determined using, for example, an enzyme immunoassay, as described, for example, in U.S. Patent Nos. 6,221,625, 5,747,273 and 5,290,708 or any another assay known in the art to determine protein concentration. When provided here, the FCDP molar concentration is determined based on the molecular weight of the FCDP dimer (eg, FCDP-BB; PM = approximately 25 KDa).

The implant methods and materials of the invention can be used to heal bone wounds of mammals, for example, fractures, implant recipient sites, and periodontal disease sites. Implant materials promote the growth of connective tissue and repair and improve bone formation compared to natural healing (that is, without added exogenous agents) or healing complemented by the addition of systemic antibiotics. Unlike natural healing, conventional surgical therapy or antibiotics, implant materials of the invention cause increased bone formation, connective tissue (e.g., cartilage or ligament) and cement when applied to damaged or diseased tissues or sites. affected by periodontal disease. The repair of these tissues leads to a better prognosis for the affected areas. The ability of these factors to stimulate the formation of new bones makes them applicable in the treatment of bone defects caused by other types of infection or surgical or accidental trauma.

Other features and advantages of the invention will be apparent from the following description of the embodiments thereof, and from the claims.

Brief description of the drawings

Figs. 1A-1G are photomicrographs that show the effect on bone formation 8 weeks after treatment. Fig. 1A is a photomicrograph showing the effect of surgery alone on bone formation. Fig. 1B is a photomicrograph showing the effect of -TCP only on bone formation. Fig. 1C is a photomicrograph showing the effect of -TCP + 0.3 mg / mL FCDP on bone formation. Fig. 1D is a photomicrograph that shows the effect of -TCP + 1.0 mg / mL FCDP on bone formation. Fig. 1E is a photomicrograph showing the effect of demineralized lyophilized bone graft (IOLD) only in bone formation. Fig. 1F is a photomicrograph showing the effect of demineralized lyophilized bone graft (IOLD) + 0.3 mg / mL FCDP in bone formation. Fig. 1G is a photomicrograph that shows the effect of demineralized lyophilized bone graft (IOLD) + 1.0 mg / mL FCDP in bone formation.

Figs. 2A-2C are photomicrographs that show the effect on bone formation 16 weeks after treatment. Fig. 2A is a photomicrograph showing the effect of -TCP only on bone formation. Fig. 2B is a photomicrograph showing the effect of -TCP + 0.3 mg / mL FCDP on bone formation. Fig. 2C is a photomicrograph showing the effect of -TCP + 1.0 mg / mL FCDP on bone formation.

Detailed description

We now describe several embodiments of the invention. Two examples demonstrating the use of FCDP as a bone and periodontium healing agent are presented below.

EXAMPLES

Example I: Preparation of FCDP

Bone wounds are treated, for example, after periodontal disease or trauma, and the periodontium, including bone, cement and connective tissue, is regenerated, according to the invention by partially combining partially purified or purified FCDP with any of the pharmaceutically transporting substances. Acceptable described above. The purified FCDP can be obtained from a recombinant source of human platelets. Commercially available recombinant FCDP can be obtained from R&amp;D System Inc. (Minneapolis, MN), BD Biosciences (San Jose, CA) and Chemicon, International (Temecula, CA). Partially purified or purified FCDP can also be prepared as follows:

Five hundred to 1000 units of washed human platelet pellets are suspended in 1M NaCl (2 ml per platelet unit) and heated at 100 ° C for 15 minutes. The supernatant is then separated by centrifugation and the precipitate is extracted twice with 1m NaCl.

The extracts are combined and dialyzed against 0.08M NaCl / 0.01M saline phosphate buffer (pH 7.4) and mixed overnight at 4 ° C with CM-Sephadex C-50 equilibrated with the buffer. The mixture is then poured into a column (5 x 100 cm), washed thoroughly with 0.08M NaCl / 0.01M sodium phosphate buffer (pH 7.4) and eluted with 1M NaCl while collecting 10 fractions. ml.

The active fractions are grouped and dialyzed against 0.3M NaCl / 0.01M sodium phosphate buffer (pH 7.4), centrifuged and passed at 4 ° C through a 2.5 x 25 cm column of blue sepharose ( Pharamacia) equilibrated with 0.3M NaCl / 0.01M phosphate buffered saline (pH 7.4). The column is then washed with the buffer and the partially purified FCDP is eluted with a 1: 1 solution of 1M NaCl and ethylene glycol.

The partially purified FCDP fractions are diluted (1: 1) with 1M NaCl, dialyzed against 1M acetic acid, and lyophilized. The lyophilized samples are dissolved in 0.8M NaCl / 0.01M sodium phosphate buffer (pH 7.4) and pass through 1.2 x 40 cm columns of CM-Shepadex C-50 equilibrated with the buffer. The FCDP is then eluted with a NaCl gradient (0.08 to 1M).

The active fractions are combined, dialyzed against 1M acetic acid, lyophilized and dissolved in a small volume of 1M acetic acid. 0.5 mL portions are applied to a 1.2 x 100 cm column of Biogel P-150 (100 to 200 mesh) equilibrated with 1M acetic acid. The FCDP is then eluted with 1M acetic acid while collecting 2 mL fractions.

Each active fraction containing 100 to 200 mg of protein is lyophilized, dissolved in 100 mL of 0.4% trifluoroacetic acid and subjected to high performance liquid chromatography in reverse phase on a Bondapak (Waters) phenyl column. Elution with a linear gradient of acetonitrile (0 to 60%) produces pure FCDP.

FCDP made by recombinant DNA technology can be prepared as follows:

The platelet-derived growth factor (FCDP) derived from human platelets contains two polypeptide sequences (FCDP-B and FCDP-A polypeptides; Antoniades, HN and Hunkapiller, M., Science 220: 963965, 1983). FCDP-B is encoded by the gene located on chromosome 7 (Betsholtz, C. et al., Nature 320: 695699) and FCDP-A is encoded by the oncogene sis (Doolittle, R. et al., Science 221: 275 -277, 1983) located on chromosome 22 (Dalla-Favera, R., Sciene 218: 686-688, 1982). This sis gene encodes the transforming protein of Simian Sarcoma Virus (VSS) that is closely related to the FCDP-2 polypeptide. Human cell C-sis also encodes the FCDP-A chain (Rao, CD, et al., Proc. Natl. Aca. Sci. USA 83: 2392-2396, 1986). Because two FCDP polypeptide chains are encoded by two different genes located on separate chromosomes, there is a possibility that human FCDP consists of heterodimer bound to disulphide of FCDP-B and FCDP-A, a mixture of the two homodimers (homodimer FCDP-BB and FCDP-AA homodimer), or a mixture of the heterodimer and the two homodimers.

Cultured mammalian cells infected with Simian Sarcoma Virus, which contains the gene that encodes the FCDP-A chain, were shown to synthesize the FCDP-A polypeptide and process it in a disulfide-bound homodimer (Robbins et al., Nature 305: 505-608, 1983). In addition, the FCDP-A homodimer reacts with elevated antiserum against human FCDP. In addition, the functional properties of the secreted FCDP-A homodimer are similar to those of platelet-derived FCDP in that it stimulates DNA synthesis in cultured fibroblasts, induces phosphorylation in the tyrosine residue of a 185 kD cell membrane protein and that It is capable of competing with (125I) -FCDP human to bind to specific cell surface FCDP receptors (Owen A. et al., Science 225: 54-56, 1984). Similar properties are shown for sis / FCDP-A gene product derived from normal cultured human cells (e.g., human arterial endothelial cells) or from human malignant cells expressing the sis / FCDP-2 gene (Antoniades, H. et al. , Cancer Cells 3: 145-151, 1985).

The FCDP-B recombinant homodimer is obtained by introducing c-sis / FCDP-B cDNA clones into mouse cells using an expression vector. The c-sis / FCDP-B clone used for expression was obtained from cultured normal human endothelial cells (Collins, T., et al., Nature 216: 748-750, 1985).

FCDP use

FCDP alone or in combination with other growth factors is useful for promoting bone healing, bone growth and regeneration or healing of the supporting structures of injured teeth due to trauma or disease. It is also useful for promoting the healing of a tooth extraction site, for increased mandibular edge, or dental implant sites. Bone healing would also be improved at the site of bone fracture or infected area, for example, osteomyelitis or tumor site. FCDP is also useful for promoting the growth and healing of a ligament, for example, the periodontal and cement ligament.

In practice, FCDP or other growth or differentiation factor is applied directly to the area that needs healing or regeneration. Generally, it is applied to a resorbable or non-absorbable carrier as a liquid

or solid, and the site is then covered with a bandage or nearby tissue. A sufficient amount to promote bone growth is generally between 500 ng and 5 mg for an area of ​​1 cm2, but the upper limit is actually 1 mg for an area of ​​1 cm2 with a preferred amount of FCDP applied 0.3 mg / mL

Example II: Periodontal regeneration with osteoconductive frameworks treated with hrFCDP-BB

The effectiveness of FCDP in promoting the growth of periodontium and bone is demonstrated with the following study.

Dog study in vivo

The beagle dog is the most used animal model to test putative periodontal regeneration materials and procedures (Wikesjo et al., J. Clin. Periodontol. 15: 73-78, 1988; Wikesjo et al., J. Clin. Periodontol. 16 : 116119, 1999; Cho et al., J. Periodontol. 66: 522-530, 1995; Giannobile eta l., J. Periodontol. 69: 129-137, 1998; and Clergeau et al., J. Periodontol. 67 : 140-149, 1996). The accumulation of plaque and tartar can induce gingival inflammation that can lead to marginal bone loss and the etiology of periodontitis in bones and humans can be compared. In contrast, in the disease that occurs naturally, there is a lack of uniformity between defects. In addition, as more attention has been given to oral health in colonies of canine breeders, it has become impracticable to obtain animals with natural periodontal disease. Therefore, the surgically induced horizontal class II furcation model has become one of the most commonly used models to investigate periodontal healing and regeneration.

Beagle dogs with horizontal Class II furcation defects were treated using FCDP compositions of the invention. Fifteen adult beagle dogs contributed 60 treated defects. Forty-two defects underwent biopsy two months after treatment and fifteen defects underwent biopsy four months after treatment.

Defect Preparation

The “critical size” periodontal defect model was used as numerous researchers describe it (see, for example, Wikesjo, 1988 and 1999, supra; Giannobile, supra, Cho, supra and Park et al., J. Periodontol. 66: 462477, 1995). Both mandibular quadrants were used in 16 male beagle dogs (2-3 years of age) without general and oral health problems. One month before the dose, the animals were sedated with a subcutaneous injection of atropine (0.02 mg / kg) and acepromazine (0.2 mg / kg) approximately 30 minutes before being anesthetized with an IV injection of sodium pentobarbital ( 25 mg / kg) After local infiltration of the surgical area with Lidocaine HCl plus epinephrine 1: 100,000, full-thickness mucoperiosteal flaps were reflected and the first and third premolar (P1 and P3) were removed. In addition, the mesial part of the crown of the first molar was removed.

The alveolar bone is then removed around the full circumference of P2 and P3, including the furcation area using chisels and water-cooled carbide and diamond drills. Horizontal bone defects were created in such a way that there was a distance of 5 mm from the fornix of the bifurcation to the bone crest. The defects were approximately 1 cm wide, depending on the width of the tooth. The roots of all experimental teeth were cleaned with curettes and ultrasonic instruments and treated with instruments with a narrow diamond drill to remove cement. After creating the standardized bone defects, the gum flaps were sutured to achieve a main closure. The animals were fed a soft diet and received daily chlorhexidine rinses for the duration of the study.

Application of graft material

Periodontal defects of P2 and P2 in each mandibular quadrant of the 15 animals were randomized before treatment using sealed envelopes. Approximately four weeks after the preparation of the defect, the animals were again anesthetized as described above and full-thickness flaps were reflected in both mandibular quadrants. A notch was placed on the tooth root surfaces in the residual bone crest using a semicircular burr to serve as a future histological reference point. The sites were irrigated with sterile saline and the roots were treated with citric acid as previously described in order to decontaminate and remove the sample layer (See, for example, Cho, supra and Park, supra). During this period a sufficient amount of -TCP or DFDBA was saturated to fill the periodontal defect with a solution of FCDP-BBhr solution (0.3 to 1.0 mg / ml) and the FCDPhr-BB / graft mixture was allowed to settle on a sterile surgical base for approximately ten minutes. The saturated FCDPhr graft was then packed in the defect with gentle pressure to the ideal level of bone regeneration.

After implantation of the graft material, the mucoperiosteal flaps were sutured approximately to the level of the enamel cement joint (ECU) using interproximal sutures, of interrupted 4.0 expanded polytetrafluoroethylene (PTFEe). After suturing the flaps, chlorhexidine gluconate gel was gently placed around the teeth and gums.

Treatment and control groups

Defects received:

<dl><dt>1.</dt><dd> -TCP </dd></dl>

<dl><dt>2.</dt><dd> -TCP plus FCDP-BBhr (0.3 mg / ml FCDPhr-BB) </dd></dl>

<dl><dt>3.</dt><dd> -TCP plus FCDP-BBhr (1.0 mg / ml FCDPhr-BB) </dd></dl>

<dl><dt>4.</dt><dd> DFDBA in dog </dd></dl>

<dl><dt>5.</dt><dd> DFDBA in dogs plus FCDP-BBhr (0.3 mg / ml FCDPhr-BB) </dd></dl>

<dl><dt>6.</dt><dd> DFDBA in dogs plus FCDP-BBhr (1.0 mg / ml FCDPhr-BB) </dd></dl>

<dl><dt>7.</dt><dd> Simulation surgery (treated only by open flap debridement, without graft) </dd></dl>

Six defects per treatment group underwent biopsy at two months (42 total sites). In addition, five defects in treatment groups 1, 2 and 3 underwent biopsy at four months (15 total sites).

Table 2. Experimental design

<dl><dt>GROUP Nº </dt><dd>NUMBER OF TEST SITES TREATMENT TEMPORARY POINTS </dd></dl>

<dl><dt>1 </dt><dd>eleven -TCP only 8 and 16 weeks n = 6 for 8 weeks n = 5 for 16 weeks </dd></dl>

<dl><dt>2 </dt><dd>eleven -TCP + 0.3 mg / ml FCDPhr-BB 8 and 16 weeks n = 6 for 8 weeks n = 5 for 16 weeks </dd></dl>

<dl><dt>3 </dt><dd>eleven -TCP + 1.0 mg / ml FCDPhr-BB 8 and 16 weeks n = 6 for 8 weeks n = 5 for 16 weeks </dd></dl>

<dl><dt>4 </dt><dd>6 DFDBA alone 8 weeks </dd></dl>

<dl><dt>5 </dt><dd>6 DFDBA + 0.3 mg / ml FCDPhr-BB 8 weeks </dd></dl>

<dl><dt>6 </dt>&lt;dd /&gt;<dt>6 </dt><dd>DFDBA + 1.0 mg / ml FCDPhr-BB 8 weeks </dd></dl>

<dl><dt>7 </dt><dd>6 Surgery, without graft 8 weeks </dd></dl>

Therefore, at 8 weeks there are 7 groups divided between 42 sites in 11 dogs. At 16 weeks, there are 3 groups divided into 15 sites into 4 dogs (one dog received staggered treatment surgeries eight weeks later and thus contributed two sites to each of the 8 and 16 week time points).

Post-surgical treatment

The surgical sites were protected by feeding the dogs on a soft diet during the first 4 weeks after the operation. To ensure optimal cure, systemic antibiotic treatment with penicillin G benzathine was provided during the first two weeks and plaque control was maintained by daily irrigation with 2% chlorhexidine gluconate throughout the experiment. The sutures were removed after 3 weeks.

Data Collect

Basics for data collection points

The 8-week time point was chosen because it is the most common time point presented for this model in the literature and therefore there are substantial historical data. For example, Wikeskjo et al., Supra, and Giannobile et al., Supra, also chose 8 weeks to evaluate the regenerative effect of PMO-2 and OP-1, respectively, in the same model. In addition, Park et al., Supra, evaluated the effect of FCDP-BBhraplicated directly to the conditioned root surface and without the GTR membranes in the beagle dog model at 8 weeks. These studies strongly suggest that the 8-week period should be optimal to illustrate the potential significant effects among various treatment modalities.

The 16-week time point was chosen to assess the long-term effects of growth factor treatment. Previous studies (Park et al., Supra) suggest that, at this time, there is substantial spontaneous healing of bone defects. However, it is possible to assess whether FCDP-BBhr treatment leads to an unusual or abnormal tissue response, such as altered bone remodeling, tumorigenesis or root resorption.

Biopsies and treatment evaluations

At the time of the biopsy, the animals were given a perfusion with 4% paraformaldehyde and sacrificed. Then, the jaws were removed and placed in a fixative. Periapical radiographs were taken and the treated sites were cut into individual blocks using a diamond saw. The coded blocks (blind) were wrapped in gauze, immersed in a 4% formaldehyde solution, processed and analyzed.

During the processing the biopsies were dehydrated in ethanol and infiltrated and embedded in methyl methacrylate. Decalcified sections of approximately 300 μm thick were obtained using a low speed diamond saw with coolant. The sections were glued in opalescent acrylic glass, crushed to a final thickness of approximately 80 μm and stained with toluidine blue and basic fuchsia. Serial sections of the stages were obtained in a mesiodistal plane.

Histomorphometric analyzes were performed on masked slides. The following parameters were evaluated:

<dl><dt>1.</dt><dd> Length of the New Union Complete Apparatus (CNAA): Periodontal regeneration measured as the distance between the coronal level of the old bone and the coronal level of the new bone, including only that new bone adjacent to the new cement with a functionally oriented periodontal ligament between the new bone and the new cement. </dd></dl>

<dl><dt>2.</dt><dd> New Bone Filler (HN): Measured as the cross-sectional area of ​​new bone formed within the furcation. </dd></dl>

<dl><dt>3.</dt><dd> Connective Tissue Filling (CT): Measured as the area within the furcation occupied by the gingival connective tissue. </dd></dl>

<dl><dt>4.</dt><dd> Vacuum (VA): The recession area where there is absence of tissue </dd></dl>

Results

A. Clinical observations

Clinically, all sites healed well. There was an impression that the sites treated with FCDP-BBhrse healed faster, as indicated by the presence of a firm and pink gum at one week post-operative. No adverse events were experienced in any treatment group, such as when evaluated by visual inspection of the treated sites. There appeared to be a greater gingival recession in groups that received -TCP or DFDBA.

B. radiographic observations

Radiographically, there was evidence of greater bone formation at two months as judged by the greater radiopacity in Groups 2, 3 ( -TCP + FCDP-BBhr0.3 and 1.0 mg / ml, respectively) and 6 (DFDBA + FCDPBBhr1.0 mg / ml) compared to the other groups (Figures 1A-G). At four months, there was evidence of increased bone formation in all groups compared to the two month time point. There was no radiographic evidence of any abnormal bone remodeling, root resorption or ankylosis in any group.

Table 3. Radiographic results. Sort order

<dl><dt>QUALITATIVE EVALUATION OF BONE FILLING AT 8 WEEKS * </dt><dd>TREATMENT </dd></dl>

<dl><dt>6 </dt><dd>-TCP only </dd></dl>

<dl><dt>4 </dt><dd>-TCP + 0.3 mg / ml FCDPhr </dd></dl>

<dl><dt>2 </dt><dd>-TCP + 1.0 mg / ml FCDPhr </dd></dl>

<dl><dt>7 </dt><dd>DFDBA alone </dd></dl>

<dl><dt>5 </dt><dd>DFDBA + 0.3 mg / ml FCDPhr </dd></dl>

<dl><dt>3 </dt><dd>DFDBA + 1.0 mg / ml FCDPhr </dd></dl>

<dl><dt>4 </dt><dd>Surgery, without graft </dd></dl>

* 1 = more padding; 7 = less padding

C. Histomorphometric analysis

The histomorphometric assessment of the length of new cement, new bone and new periodontal ligament (CNAA) as well as new bone filling, connective tissue filling and empty space was evaluated and expressed as percentages. In the case of CNAA, the values ​​for each test group represent the CNAA measurements (length in m / m) / total available CNAA length (in mm) x 100%. Bone filling, connective tissue filling and empty space were evaluated and expressed as percentages of the total furcation defect area.

The single-factor variance analysis (ANOVA) was used to test the general differences between treatment groups, and comparisons were made in pairs using the student's t-test. Significant differences were found between groups after analysis of the coded slides. Table 4 shows the results at two months.

Table 4. Histometric analysis at two months

<dl><dt>GROUP Nº </dt><dd>TREATMENT % PERIODONTAL REGENERATION CNAA % BONE FILLING % FILLING CONNECTIVE FABRIC % EMPTY </dd></dl>

<dl><dt>1 </dt><dd>-TCP only 37.0 ± 22.8 ** 28.0 ± 29.5 36.0 ± 21.5 12.0 ± 17.9 </dd></dl>

<dl><dt>2 </dt><dd>-TCP + 0.3 mg / ml FCDPhr 59.0 ± 19.1 *, Ɩ 84.0 ± 35.8 0.0 ± 0.0 8.0 ± 17.9 </dd></dl>

<dl><dt>3 </dt><dd>-TCP + 1.0 mg / ml FCDPhr 46.0 ± 12.3 * 74.2 ± 31.7 ƖƖ 0.0 ± 0.0 0.0 ± 0.0 </dd></dl>

<dl><dt>4 </dt><dd>DFDBA alone 13.4 ± 12.0 6.0 ± 8.9 26.0 ± 19.5 30.0 ± 27.4 </dd></dl>

<dl><dt>5 </dt><dd>DFDBA + 0.3 mg / ml FCDPhr 21.5 ± 13.3 20.0 ± 18.7 36.0 ± 13.4 18.0 ± 21.7 </dd></dl>

<dl><dt>6 </dt><dd>DFDBA + 1.0 mg / ml FCDPhr 29.9 ± 12.4 46.0 ± 23.0 f 26.0 ± 5.48 8.0 ± 13.04 </dd></dl>

<dl><dt>7 </dt><dd>Surgery simulation, without grafting 27.4 ± 15.0 34.0 ± 27.0 48.0 ± 35.64 10.0 ± 22.4 </dd></dl>

* Groups 2 and 3 significantly larger (p &lt;0.05) than Groups 4 and 7 ** Group 1 significantly higher (p &lt;0.05) than Group 4 t Group 2 significantly higher (p &lt;0.05) than Group 5 tt Groups 2 and 3 significantly larger than Groups 1, 4 and 7 f Group 6 significantly larger than Group 4

The average percentage periodontal regeneration (CNAA) in surgery groups without grafts plus surgery more

-TCP was only 27% and 37%, respectively. However, the -TCP groups containing FCDP-BBhr showed significantly more periodontal regeneration (p &lt;0.05) than graft surgery or DFDBA alone (59% and 46% respectively for concentrations 0.3 and 1.0 mg / ml versus 27% for surgery alone and 13% for DFDBA alone). Finally, the -TCP group containing 0.3 mg / ml FCDP-BBhr showed significantly more periodontal regeneration (p&gt; 0.05) than the same concentration of FCDP-BBhr combined with graft (59% vs. 21% ).

Bone filling was significantly higher (p&gt; 0.0.5) in the groups of -TCP + 0.3 mg / ml FCDP-BBhr (84.0%) and -TCP + 1.0 mg / ml FCDP -BBhr (74.2%) than in the treatment groups of -TCP alone (28.0%), surgery alone (34%) or DFDBA alone (6%). There was also significantly greater bone filling (p&gt; 0.05) for the -TCP + 0.3 mg / ml FCDP-BBhr group compared to the DFDBA + 0.3 mg / ml FCDP-BBhr group (84% and 20% respectively).

The analysis group that examined the 8-week data from the DFDBA groups and the surgery group alone (Groups 4, 5, 6 and 7) did not show statistically significant difference between the DFDBA groups and surgery only for periodontal regeneration (CNNA). There was a tendency for greater regeneration for those sites treated with DFDBA improved with 1.0 mg / ml FCDP-BBhr against DFDBA alone. There was a significantly improved bone filling (p&gt; 0.05) for sites treated with DFDBA + 1.0 mg / ml FCDP-BBhr than DFDBA alone (46 and 6% respectively). There was a tendency for greater bone filling for sites treated with DBDBA containing 0.3 mg / ml FCDP-BBhr compared to DFDBA alone or surgery alone. However, sites treated with DFDBA only demonstrated less bone filling in the defect than surgery alone (6 and 34%, respectively), missing most of the defect of any filling or filling consisting of gingival connective tissue (soft).

Four months after treatment, there were significant differences in periodontal regeneration. -TCP alone, as a result of extensive ankylosis, resulted in 36% regeneration, while sites treated with -TCP containing FCDP-BBhr had an average regeneration of 58% and 49% at concentrations of 0, 3 and 1.0 mg / ml FCDPhr-BB. There was a substantial bone filling in the three treatment groups. -TCP only resulted in 70% bone filling, -TCP plus 0.3 mg / ml FCDP produced 100% bone filling while the 1.0 mg / ml FCDP group had 75% filling.

D. Histological Evaluation

Histological evaluation was performed for all biopsies except one, in which the evaluation was not possible due to difficulties encountered during processing.

Representative photomicrographs are shown in Figures 1A-G and 2A-C. Figure 1A shows results of a site treated only with surgery (without grafts). This specimen demonstrates limited periodontal regeneration (new bone (HN), new cement (CN), and periodontal ligament (LP)) as demonstrated in the area of ​​the notches and which extends only a short distance coronally. The furcation area is mainly occupied by dense soft connective tissue (CT) with minimal formation of new bone (HN).

For sites treated with -TCP alone (Figure 1B), there is periodontal regeneration, similar to that observed for the surgical specimen alone, which extends from the base of the notches for a coronally short distance. As seen in the surgery specimen alone, there was very little new bone formation with the largest area of ​​the furcation occupied by soft connective tissue.

However, Figure 1C illustrates the results obtained for sites treated with -TCP + 0.3 mg / ml FCDPhr-BB. A significant periodontal regeneration is shown with new bone, new cement and periodontal ligament that extends along the entire surface of the furcation. In addition, the furcation area is full of new bone that extends the entire height of the furcation to the fornix.

Representative results for sites treated with -TCP + 1.0 mg / ml FCDPhr-BB are shown in Figure 1D. While there is significant periodontal regeneration in the furcation, it does not extend along the entire surface of the furcation. There is new bone formation present along the soft connective tissue that is observed in the coronal part of the defect along with a small space that is empty of any tissue (VA) in the furnace fornix.

Figures 2A, 2B and 2C illustrate results obtained for the groups with graft treatment. The representative results for the DFDBA group alone (Figure 2A) show a poor periodontal regeneration that is limited to the area of ​​the notch that extends only slightly in a coronal direction. New bone formation is limited and consists of small amounts of bone formation along the surface of residual DFDBA graft material (dark red dye along lighter pink islands). In addition, the new bone is surrounded by extensive soft connective tissue that extends coronally to fill a significant area in the furcation. Finally, a large empty space extends from the coronal extension of the soft connective tissue to the fornix of the furcation.

Histological results for DFDBA + 0.3 and 1.0 mg / ml FCDP-BBhrse are shown in Figures 2B and 2C, respectively. Both groups demonstrate greater periodontal regeneration compared to DFDBA only with a new attachment apparatus (new bone, new cement and new periodontal ligament) that extends from the base of the notches in the roots for a short distance coronally (arrows9. They also had a greater bone filling in the furcation area, although there was a significant filling of the furcation with soft connective tissue.

Conclusions

Based on the results of the study, the treatment of a periodontal defect using FCDP-BBhr at 0.3 mg / mL or 1.0 mg / mL in combination with a suitable carrier material (e.g., -TCP) results in resulted in greater periodontal regeneration than current products or procedures, such as grafts with

-TCP or bone graft alone, or periodontal surgery without grafts.

Treatment with concentrations 0.3 mg / mL and 1.0 mg / mL of FCDPhr resulted in periodontal regeneration. The 0.3 ml / mL concentration of FCDPhr demonstrated a higher periodontal regeneration and bone filling percentage compared to the 1.0 mg / ml concentration of FCDPhr when mixed with -TCP.

-TCP was more effective than grafting when mixed with FCDP-BBhr at any concentration. The new bone matured (remodeled) normally with the passage of time (0, 8 and 16 weeks) in all groups. There was no increase in ankylosis or root reabsorption in the FCDPhr groups. In fact, sites that received FCDP-BBhr tended to have less ankylosis than control sites. This finding may be the result of the fact that FCDP-BBhr is mitogenic and chemotactic for periodontal ligament cells.

MATERIALS AND METHODS

Materials used: Test and control articles

The -TCP used a particle size (0.25 mm - 1.0 mm) that was optimized for periodontal use. Based on studies using a canine model, the administered -TCP is reabsorbed ~ 80% in three months and is replaced by autologous bone during the healing process.

The DFDBA was provided by the Musculoskeletal Transplant Foundation (FTM). The material was dog allograft, made of bones of a dog that was killed after the completion of another study that tested a surgical procedure that was considered to have no effect on skeletal tissues.

Recombinant FCDPh-BB was supplied by BioMimetic Pharmaceuticals and manufactured by Chiron, Inc, the only supplier of FCDP-BBhr for human use approved by AAF. The AAF approved this FCDP-BBhr as a wound healing product under the trade name Regranex®

One ml syringes containing 0.5 ml of sterile FCDP-BBhr were prepared in two concentrations in accordance with AAF standards for human materials and in accordance with the current applicable Good Manufacturing Process (APBF). The concentrations tested included 0.3 mg / ml and 1.0 mg / ml.

-TCP was provided in vials containing 0.5 cc of sterile particles.

DFDBA was provided in 2.0 ml syringes containing 1.0 ml of lyophilized, demineralized and sterile dog bone allograft.

Preparation of material

At the time of the surgical procedure, the final implanted grafts were prepared by mixing the FCDP-BBhr solution with the matrix materials. In summary, a sufficient amount of TCP or allograft to completely fill the bone defect was placed in a sterile dish. The FCDP-BBhr solution sufficient to completely soak the matrix was then added, the materials were mixed and allowed to sit on the surgical tray for approximately 10 minutes at room temperature before placing in the bone defect.

An incubation time of 10 minutes with the -TCP material is sufficient to obtain maximum adsorption of the growth factor (see Appendix A). It is also an appropriate amount of time for surgeons in a clinical context before product placement in the periodontal defect. Similarly, in a commercial market, the FCDP-BBhr and the matrix material can be supplied in separate containers in a kit and the materials can be mixed directly before placement. This kit concept would greatly simplify product life / stability considerations.

Example III Use of FCDP for the treatment of periodontal bone defects in humans

Recombinant human FCDP-BB (FCDP-BBhr) was tested for its effect on the regeneration of periodontal bone in human subjects. Two test groups were administered FCDP-BBhr at 0.3 mg / mL (Group I) or 1.0 mg / mL (Group II). FCDP-BBhr was prepared in sodium acetate buffer and administered in a beta-tricalcium phosphate vehicle ( -TCP). The control group, Group III, was administered -TCP in sodium acetate only.

The objective of the clinical study was to evaluate the safety and effectiveness of graft material comprising -TCP and FCDP-BBhr at 0.3 mg / mL or 1.0 mg / mL in the treatment of one (1) to three (3 ) intraosseous periodontal defects and to assess their regenerative capacity in bone and soft tissue.

Study design and duration of treatment

The study was a double-blind, controlled, prospective, randomized clinical trial, designed in parallel and multi-center in subjects that required surgical intervention to treat a bone defect adjacent to the natural dentition. The subjects were randomized in equal proportions to result in three (3) treatment groups of approximately 60 subjects each (180 in total). The duration of the study was six (6) months after the implantation of the study device. The study enrolled 180 subjects.

Diagnosis and main input criteria

The study admitted male and female subjects, 25-75 years of age, with advanced periodontal disease in at least one site that required surgical treatment to correct a bone defect. Other inclusion criteria included: 1) a probing pocket depth that averages 7 mm or more in the baseline visit; 2) after surgical debridement, a vertical bone defect (OD) of 4 mm or greater with at least 1 bone wall; 3) sufficient keratinized tissue to allow complete tissue coverage of the defect; and 4) radiographic basis of the defect at least 3 mm coronal to the apex of the tooth. The participation of subjects who smoked up to 1 package a day and who had teeth with Class I and II furcation was specifically allowed.

Dosage and mode of administration

All treatment kits contained 0.25 g of -TCP (an active control) and 0.5 mL of sodium acetate solution alone (Group III), 0.3 mg / mL FCDP-BBhr (Group I), or 1.0 mg / mL FCDP-BBhr (Group II):

After thorough debridement and root cleaning, the test solution was mixed with -TCP in a sterile container, so that the -TCP was completely saturated. Root surfaces were conditioned using tetracycline, EDTA or citric acid. The hydrated graft was then packaged in the bone defect and the tissue flaps were secured with interdental sutures to achieve complete coverage of the surgical site.

Effectiveness measurement

The main measure of effectiveness included the change in the level of clinical union (NUC) between the referral and six months after surgery (Group I vs. Group III). Secondary measures of effectiveness consisted of the following results: 1) linear bone growth (COL) and% bone filling (% RO) from the reference six months after surgery based on radiographic evaluations (Group I and Group II vs. Group III; 2) change in NUC between the referral and six months after surgery (Group II vs. Group II); 3) reduction of the depth of the probing pocket (PBS) between the reference and six months after surgery (Group I and Group II vs. Group III); 4) gingival recession (RG) between the referral and six months after surgery (Group I and Group II vs. Group III); 5) wound healing (CH) of the surgical site during the first three weeks after surgery (Group I and Group II vs. Group III); 6) area under the curve for the change in NUC between the reference and three (3) and six (6) months (Group I and Group II vs. Group II); 7) 95% lower bound trust (MCU) for% RO at six (6) months after surgery (Groups I, II and II vs. demineralized lyophilized bone allograft (DFDBA) as published in the literature; Parashis et al., J. Periodontol. 69: 751-758, 1998); 8) 95% MCU for linear bone growth at six

(6) months after surgery (Groups I, II and III vs. demineralized lyophilized bone allograft (DFDBA) as published in the literature; Persson et al., J. Clin. Periodontol. 27: 104-108, 2000) ; 9) 95% MCU for the change in NUC between reference and six (6) months (Groups I, II and II vs. EMDOGAIN® - PMA P930021, 1996); and 10) 95% MCU for the change in NUC between reference and six (6) months (Groups I, II and II vs. PEPGEN P-15 ™ - PMA P990033, 1999).

Statistical methods

Safety and effectiveness data were examined and summarized using descriptive statistics. Categorical measurements were shown as totals and percentages, and continuous variables were shown as means, medians, standard deviations and ranges. Statistical comparisons between the test product treatment groups (Groups I and II) and the control (Group III) were made using the chi-square test and Fisher's exact test for categorical variables and tests to analysis methods of variance (ANOVA) for continuous variables. Comparisons between treatment groups for ordinal variables were made using Cochran-Mantel-Haenszel methods. A p &lt;0.05 (unilateral) was considered to be statistically significant for NUC, COL and% RO.

The safety data were evaluated by the frequency and severity of adverse events such as those evaluated clinically and radiographically. Nor were there statistically significant differences observed in the incidence of adverse events (HA; all causes) between the three treatment groups. The safety analyzes did not identify any greater risk for the subject due to the implantation of the graft material.

Summary of effectiveness results

The results of the statistical analyzes revealed clinically and statistically significant benefits for the two treatment groups (Groups I and II) compared to the active control of -TCP alone (Group III) and the historical controls that include DFDBA, EMDOGAIN® and PEPGEN P-15 ™.

Three months after surgery, a statistically significant increase in NUC was observed from the referral in favor of Group I versus Group III (P = 0.041), indicating that there are significantly early benefits of FCDP in the increase in NUC. At six months after surgery, this trend continued in favor of Group I over Group III, although this difference was not statistically significant (p = 0.200). The analysis of the area under the curve (ABC) representing the cumulative effect (that is, speed) for the increase in NUC between the reference and six months approached a statistical significance in favor of Group I compared to Group III ( p = 0.054). In addition, the 95% analysis of lower bound confidence (MCU) for all treatment groups confirmed the effectiveness of Groups I and II compared to increases in NUC observed at six (6) months for EMDOGAIN® and PEPGEN P- 15 ™.

In addition to the clinical benefits observed for NUC, radiographic analyzes that include Linear Bone Growth (COL) and Bone Filler Percentage (% RO), revealed a significantly improved improvement in bone augmentation for Groups I and II vs. Group III % RO was defined as the percentage of the original bone defect filled with new bone as measured radiographically. COL showed a significant improvement in Group I (2.5 mm) when compared to Group III (0.9 mm, p &lt;0.001). COL was also significant for Group II (1.5 mm) when compared to Group III (p = 0.021).

Bone Filler Percentage (% RO) increased significantly six months after surgery in Group I (56%) and Group II (34%) when compared to Group III (18%), for a p &lt;0.001 and p = 0.019, respectively. The 95% lower confidence bound in the six-month interval after surgery, for linear bone growth and% bone filling confirmed the effectiveness of Groups I and II compared to the radiographic results published for DFDBA, the most widely material Used for periodontal grafting procedures.

At three months, there was significantly less Gingival Recession (RG) (p = 0.041) for Group I compared to Group III consistent with the beneficial effect observed with NUC. No statistically significant differences were observed in PDR and RG at six months. Descriptive analyzes of the number of sites that show complete wound healing (CH) at three months revealed improvements in Group I (72%) vs. Group II (60%) and Group III (55%), which indicates a trend towards better healing.

To assess the cumulative beneficial effect for clinical and radiographic results, a composite analysis of effectiveness was performed to determine the percentage of patients with a good outcome as defined by NUC&gt; 2.7 mm and COL&gt; 1.1 mm at six (6 ) months. The successful NUC and COL benchmarks were established for these parameters using implanted grafts, as identified in the previous section “Effectiveness measures”. The results showed that 61.7% of Group I patients and 37.9% of Group II patients met or exceeded the composite reference point for existence compared to 30.4% of Group III patients, resulting in a statistically significant benefit of Group I vs. Group II (p &lt;0.001). % RO revealed similar benefits for Group I (70.0%) vs. Group III (44.6) for the p-value of 0.003.

In summary, Group I achieved statistically beneficial results for NUC and RG at three (3) months as well as COL and% RO at six (6) months, compared to the active control group of -TCP alone (Group III). The clinical significance of these results is further confirmed by comparison with historical controls. It is concluded that the graft material containing FCDP was shown to achieve clinical and radiographic effectiveness for six months by treating periodontal bone defects.

Table 5. Summary of FCDP graft effectiveness

<dl><dt>FINAL POINT </dt><dd>GROUP I GROUP II GROUP III </dd></dl>

<dl><dt>Increase in NUC (mm): 3 months </dt><dd>3.8 (p = 0.04) 3.4 (p = 0.40) 3.3 </dd></dl>

<dl><dt>NUC: ABC analysis (mm x sem) </dt><dd>67.5 (p = 0.05) 61.8 (p = 0.35) 60.1 </dd></dl>

<dl><dt>NUC (mm): 95% MCU 6 months (vs 2.7 mm for EMDOGAIN &amp; 1 mm for PEPGEN) </dt><dd> 3,3 3,2 3,1 </dd></dl>

<dl><dt>RG (mm): 3 months </dt><dd>0.5 (p = 0.04) 0.7 (p = 0.46) 0.9 </dd></dl>

<dl><dt>COL (mm): 6 months </dt><dd>2.5 (p &lt;0.001) 1.5 (p = 0.02) 0.9 </dd></dl>

<dl><dt>% RO: 6 months </dt><dd>56.0 (p &lt;0.001) 33.9 (p = 0.02) 17.9 </dd></dl>

<dl><dt>Compound Analysis (% Success) </dt><dd>NUC-COL 61.7% (p &lt;0.001) 37.9% (p = 0.20) 30.4% </dd></dl>

<dl><dt>NUC-% RO </dt>&lt;dd /&gt;<dt>NUC-% RO </dt><dd>70.0% (p = 0.003) 44.6% </dd></dl>

The graft material (i.e., -TCP) containing FCDP at 0.3 mg / mL and 1.0 mg / mL proved safe and effective in restoring alveolar bone and clinical attachment around teeth with moderate periodontitis Advanced in a large randomized clinical trial that included 180 subjects studied for 6 months. These conclusions are based on validated radiographic and clinical measurements as summarized above.

Consistent with the biocompatibility data of the graft material containing FCDP, discussed above, and the safe historical use of each individual component (i.e., -TCP alone or FCDP alone), the study revealed no evidence of its local adverse effects or systemic. There were no adverse results attributable to the graft material, which proved to be safe.

conclusion

The implantation of FCDP containing -TCP at 0.3 mg / mL or 1.0 mg / mL proved to be an effective treatment for restoration of soft tissue and bone junction level as evidenced by the significantly improved NUC at 3 months compared to active control. Our findings were also consistent with the ABC analysis that showed an improvement in the increase in NUC between the reference and six months. The implantation of FCDP containing -TCP at 0.3 mg / mL or 1.0 mg / mL also proved to be an effective treatment based on a significantly improved COL and% CO compared to the active control. The significantly improved clinical results demonstrated by the composite analysis of soft and hard tissue measurements compared to the active control of -TCP alone also demonstrated the effectiveness of the treatment protocol described above. Finally, the results of the administration of FCDP containing -TCP at 0.3 mg / mL

or 1.0 mg / mL were found to exceed established benchmarks of effectiveness both clinically and radiographically.

The results of this trial together with extensive and confirmatory data from studies with animals and humans in vivo demonstrates that graft material containing FCDP stimulate soft and hard tissue regeneration in periodontal defects, although the effects were more significant when FCDP in the range of 0.1 to 1.0 mg / mL (for example, 0.1 mg / mL, 0.3 mg / mL or 1.0 mg / mL) was administered in the graft material. In addition, FCDP administered in the graft material in the amount of 0.3 mg / mL significantly regenerated soft tissue and bone.

Further embodiments of the present disclosure are described below.

<dl><dt>1.</dt><dd> A method for promoting the growth of bone, periodontium, ligament or cartilage of a mammal that comprises administering to said mammal an implant material comprising a platelet-derived growth factor (FCDP) at a concentration in the range of about 0 , 1 mg / mL to about 1.0 mg / mL in a pharmaceutically acceptable liquid carrier and a pharmaceutically acceptable solid carrier, where said implant material promotes the growth of said bone, periodontium, ligament or cartilage. </dd></dl>

<dl><dt>2.</dt><dd> The method of embodiment 1, wherein said FCDP has a concentration of approximately 0.3 mg / mL. </dd></dl>

<dl><dt>3.</dt><dd> The method of embodiment 2, wherein said FCDP has a concentration of 0.3 mg / mL. </dd></dl>

<dl><dt>4.</dt><dd> The method of embodiment 1, wherein said pharmaceutically acceptable solid carrier comprises one of the following: a biocompatible binder, a bone substitute agent or a gel. </dd></dl>

<dl><dt>5.</dt><dd> The method of embodiment 4, wherein said biocompatible binder is a natural or synthetic polymer. </dd></dl>

<dl><dt>6.</dt><dd> The method of embodiment 5, wherein said natural or synthetic polymer is selected from polysaccharides, nucleic acids, carbohydrates, proteins, polypeptides, collagen, poly (a-hydroxy acids), poly (lactones), poly (amino acids), poi (anhydrides ), poly (orthoesters), poly (anhydride-co-imides), poly (orthocarbonates), poly (ahydroxy alkanoates), poly (dioxanones), poly (phosphoesters), polylactic acid, poly (L-lactide) (PLLA), poly (D, Lactide), (PDLLA), polyglycolic acid, polyglycolide (PGA), poly (lactide-co-glycolide (PLGA), poly (L-lactide-co-D, L-lactide), poly (D, L-lactide-co-carbonate trimethylene), polyhydroxybutyrate (PHB), poly (£ -caprolactone ), poly (5-valerolactone), poly (y-butyrolactone), poly (caprolactone), polyacrylic acid, polycarboxylic acid, poly (allylamine hydrochloride), poly (diallyldimethylammonium chloride), poly (ethyleneimine), polypropylene fumarate, polypropylene alcohol polyvinyl, polyvinylpyrrolidione, poly (ethyloxazoline), block copolymers poly (ethylene oxide) -co-poly (propylene oxide), poly (ethylene teraphthalate) polyamide, and copolymers and mixtures thereof. </dd></dl>

<dl><dt>7.</dt><dd> The method of embodiment 5, wherein said natural or synthetic polymer is selected from collagen, polyglycolic acid, polylactic acid and polymethylmethacrylate. </dd></dl>

<dl><dt>8.</dt><dd> The method of embodiment 4, wherein said biocompatible binder is selected from alginic acid, gum arabic, guar gum, xanthan gum, gelatin, chitin, chitosan, chitosan acetate, chitosan lactate, chondroitin sulfate, NO-carboxymethyl chitosan, a dextran, fibrin glue, glycerol, hyaluronic acid, sodium hyaluronate, a cellulose, a glucosamine, a proteoglycan, a starch, lactic acid, a pluronic, sodium glycerophosphate, collagen, glycogen, a keratin, silk and derivatives and mixtures thereof. </dd></dl>

<dl><dt>9.</dt><dd> The method of embodiment 4, wherein said biocompatible binder is sodium hyaluronate or derivatives thereof. </dd></dl>

<dl><dt>10.</dt><dd> The method of embodiment 9, wherein said biocompatible binder is hyaluronic acid. </dd></dl>

<dl><dt>11.</dt><dd> The method of embodiment 4, wherein said biocompatible binder is selected from methyl cellulose, carboxymethyl cellulose, hydroxypropyl methylcellulose or hydroxyethyl cellulose. </dd></dl>

<dl><dt>12.</dt><dd> The method of embodiment 10, wherein said biocompatible binder is carboxymethyl cellulose. </dd></dl>

<dl><dt>13.</dt><dd> The method of embodiment 8, wherein said dextran is a-cyclodextrin, -cyclodextrin, and -cyclodextrin, or sodium dextran sulfate. </dd></dl>

<dl><dt>14.</dt><dd> The method of embodiment 8, wherein said starch is hydroxyethyl starch or soluble starch. </dd></dl>

<dl><dt>15.</dt><dd> The method of embodiment 4, wherein said bone substitute agent is selected from calcium phosphate, calcium sulfate or demineralized bone. </dd></dl>

<dl><dt>16.</dt><dd> The method of embodiment 15, wherein said calcium phosphate is selected from tricalcium phosphate, hydroxyapatite, low crystalline hydroxyapatite, amorphous calcium phosphate, calcium metaphosphate, dicalcium phosphate dihydrate, heptacalcium phosphate, calcium pyrophosphate dihydrate, calcium pyrophosphate and calcium phosphate. </dd></dl>

<dl><dt>17.</dt><dd> The method of embodiment 15, wherein said calcium phosphate is provided as a paste or putty that forms a hardened calcium phosphate after in vivo administration. </dd></dl>

<dl><dt>18.</dt><dd> The method of embodiment 15, wherein said calcium phosphate is provided as a hardened calcium phosphate. </dd></dl>

<dl><dt>19.</dt><dd> The method of embodiment 15, wherein said calcium phosphate is bioresorbable. </dd></dl>

<dl><dt>20.</dt><dd> The method of embodiment 16, wherein said tricalcium phosphate is -tricalcium phosphate ( -TCP). </dd></dl>

<dl><dt>21.</dt><dd> The method of embodiment 20, wherein said -TCP comprises a matrix of micron-sized particles </dd></dl>

or submicron.

<dl><dt>22.</dt><dd> The method of embodiment 21, wherein said -TCP particles are smaller than 5000 μm in size. </dd></dl>

<dl><dt>23.</dt><dd> The method of embodiment 21, wherein said -TCP particles have a particle size in the range of about 100 to about 5000 µm. </dd></dl>

<dl><dt>24.</dt><dd> The method of embodiment 23, wherein said -TCP particles have a particle size in the range of about 100 to about 3000 µm. </dd></dl>

<dl><dt>25.</dt><dd> The method of embodiment 24, wherein said -TCP particles have a particle size in the </dd></dl>

range from about 250 to about 2000 μm.

<dl><dt>26.</dt><dd> The method of embodiment 21, wherein said -TCP particles are porous. </dd></dl>

<dl><dt>27.</dt><dd> The method of embodiment 26, wherein said -TCP particles have a porosity greater than 40%. </dd></dl>

<dl><dt>28.</dt><dd> The method of embodiment 27, wherein said -TCP particles have a porosity greater than 65%. </dd></dl>

<dl><dt>29.</dt><dd> The method of embodiment 28, wherein said -TCP particles have a porosity greater than 90%. </dd></dl>

<dl><dt>30.</dt><dd> The method of embodiment 20, wherein said -TCP is provided in a form suitable for implantation. </dd></dl>

<dl><dt>31.</dt><dd> The method of embodiment 30, wherein said shape is selected from a sphere, a cylinder and a block. </dd></dl>

<dl><dt>32.</dt><dd> The method of embodiment 15, wherein said demineralized bone is cortical or spongy bone. </dd></dl>

<dl><dt>33.</dt><dd> The method of embodiment 1, wherein said pharmaceutically acceptable liquid carrier is selected from gua, a physiologically acceptable buffer or a cell culture medium. </dd></dl>

<dl><dt>34.</dt><dd> The method of embodiment 33, wherein said physiologically acceptable buffer is sodium acetate buffer. </dd></dl>

<dl><dt>35.</dt><dd> The method of embodiment 1, wherein said composition further comprises a biologically active agent. </dd></dl>

<dl><dt>36.</dt><dd> The method of embodiment 35, wherein said biologically active agent is selected from an antibody, an antibiotic, a polynucleotide, a polypeptide, a protein, an anti-cancer agent, a growth factor, an anti-inflammatory agent and a vaccine. </dd></dl>

<dl><dt>37.</dt><dd> The method of embodiment 36, wherein said protein is an osteogenic protein. </dd></dl>

<dl><dt>38.</dt><dd> The method of embodiment 37, wherein said osteogenic proteins is selected from growth factor </dd></dl>

insulin I (FCI-I), insulin growth factor II (FCI-II), transforming growth factor-2 (FCT-2), transforming growth factor-a (FCT-a), a bone morphogenetic protein (PMO) or osteogenic.

<dl><dt>39.</dt><dd> The method of embodiment 1, wherein said implant material further comprises autologous bone marrow or autologous platelet extracts. </dd></dl>

<dl><dt>40.</dt><dd> The method of embodiment 1, wherein said FCDP is partially or substantially purified. </dd></dl>

<dl><dt>41.</dt><dd> The method of embodiment 1, wherein said FCDP is obtained from a natural source or a recombinant source. </dd></dl>

<dl><dt>42.</dt><dd> The method of embodiment 41, wherein said natural source comprises blood, platelets, serum, platelet concentrate, plasma-rich pasma (PRP) or bone marrow. </dd></dl>

<dl><dt>43.</dt><dd> The method of embodiment 41, wherein said natural source is platelet rich plasma (PRP). </dd></dl>

<dl><dt>44.</dt><dd> The method of embodiment 1, wherein said implant material delivers said FCDP to said bone, periodontium, ligament or cartilage for at least one day after administration. </dd></dl>

<dl><dt>45.</dt><dd> The method of embodiment 1, wherein said implant material delivers said FCDP to said bone, periodontium, ligament or cartilage for less than about 28 days after administration. </dd></dl>

<dl><dt>46.</dt><dd> The method of embodiment 1, wherein said implant material delivers said FCDP to said bone, periodontium, ligament or cartilage for less than about 21 days after administration. </dd></dl>

<dl><dt>47.</dt><dd> The method of embodiment 1, wherein said implant material delivers said FCDP to said bone, periodontium, ligament or cartilage for less than about 14 days after administration. </dd></dl>

<dl><dt>48.</dt><dd> The method of embodiment 1, wherein said implant material delivers said FCDP to said bone, periodontium, ligament or cartilage from about 1 day to about 14 days after administration. </dd></dl>

<dl><dt>49.</dt><dd> The method of embodiment 1, wherein said bone, periodontium, ligament or cartilage is damaged. </dd></dl>

<dl><dt>50.</dt><dd> The method of embodiment 1 which further comprises the step of allowing said bone, periodontium, ligament or cartilage to grow. </dd></dl>

<dl><dt>51.</dt><dd> The method of embodiment 50 further comprising the steps of exposing said bone, periodontium, ligament or cartilage producing a surgical skin flap before administering said implant material, and replacing said flap after administering said implant material. </dd></dl>

<dl><dt>52.</dt><dd> The method of embodiment 51 further comprising, after the stage of producing a surgical skin flap to expose said bone, periodontium or ligament, but before stage (a), the stage of cleaning said bone or periodontium to remove matter organic of said bone or periodontium. </dd></dl>

<dl><dt>53.</dt><dd> The method of embodiment 1, wherein said FCDP is released from the implant material after administration at an average rate of less than or equal to 300 μg / day. </dd></dl>

<dl><dt>54.</dt><dd> The method of embodiment 1, wherein said FCDP is released from the implant material after administration at an average rate of less than 100 μg / day. </dd></dl>

<dl><dt>55.</dt><dd> The method of embodiment 1, wherein said FCDP is released from the implant material after administration at an average rate of less than 50 μg / day. </dd></dl>

<dl><dt>56.</dt><dd> The method of embodiment 1, wherein said FCDP is released from the implant material after administration at an average rate of less than 10 μg / day. </dd></dl>

<dl><dt>57.</dt><dd> The method of embodiment 1, wherein said FCDP is released from the implant material after administration at an average rate of less than 1 μg / day. </dd></dl>

<dl><dt>58.</dt><dd> The method of embodiment 1, wherein said pharmaceutically acceptable carrier is sterile. </dd></dl>

<dl><dt>59.</dt><dd> The method of embodiment 1, wherein said FCDP is FCDP AA, FCDP BB, FCDP CC or FCDP DD or combinations or derivatives thereof. </dd></dl>

<dl><dt>60.</dt><dd> The method of embodiment 59, wherein said FCDP is FCDP-BB. </dd></dl>

<dl><dt>61.</dt><dd> The method of embodiment 59, wherein said FCDP is FCDP-AB. </dd></dl>

<dl><dt>62.</dt><dd> A method to promote the growth of bone, periodontium, ligament or cartilage of a mammal that </dd></dl>

comprises (a) administering to said mammal an implant material comprising platelet-derived growth factor (FCDP) in a concentration in the range of less than or equal to 0.3 mg / mL in a pharmaceutically acceptable liquid carrier and a solid carrier pharmaceutically acceptable, where said implant material promotes the growth of said bone, periodontium, ligament or cartilage.

<dl><dt>63.</dt><dd> A vial comprising platelet-derived growth factor (FCDP) in a concentration in the range of approximately 1.0 mg / mL in a pharmaceutically acceptable carrier. </dd></dl>

<dl><dt>64.</dt><dd> The vial of embodiment 63, wherein said liquid is sterile sodium acetate buffer. </dd></dl>

<dl><dt>65.</dt><dd> The vial of embodiment 63 comprising FCDP at a concentration of approximately 0.3 mg / mL. </dd></dl>

<dl><dt>66.</dt><dd> The vial of embodiment 63, wherein said FCDP is FCDP-BB. </dd></dl>

<dl><dt>67.</dt><dd> The vial of embodiment 64, wherein said FCDP is stable in said buffer for at least 36 months when stored at a temperature in the range of 2 ° C to 80 ° C. </dd></dl>

<dl><dt>68.</dt><dd> The vial of embodiment 64, wherein said FCDP is stable in said buffer for at least 24 months when stored at a temperature in the range of 2 ° C to 80 ° C. </dd></dl>

<dl><dt>69.</dt><dd> The vial of embodiment 64, wherein said FCDP is stable in said buffer for at least 18 months when stored at a temperature in the range of 2 ° C to 80 ° C. </dd></dl>

<dl><dt>70.</dt><dd> The vial of embodiment 64, wherein said FCDP is stable in said buffer for at least 12 months when stored at a temperature in the range of 2 ° C to 80 ° C. </dd></dl>

<dl><dt>71.</dt><dd> An implant material comprising porous calcium phosphate having adsorbed a liquid comprising platelet-derived growth factor (FCDP) in a concentration in the range of about 0.1 mg / mL to about 1.0 mg / mL. </dd></dl>

<dl><dt>72.</dt><dd> The implant material of embodiment 71, wherein the concentration of FCDP is approximately 0.3 mg / mL. </dd></dl>

<dl><dt>73.</dt><dd> The implant material of embodiment 71, wherein said calcium phosphate is selected from tricalcium phosphate, hydroxyapatite, low crystalline hydroxyapatite, amorphous calcium phosphate, calcium metaphosphate, dicalcium phosphate dihydrate, heptacalcium phosphate, calcium pyrophosphate dihydrate, calcium pyrophosphate and calcium phosphate octacalcium </dd></dl>

<dl><dt>74.</dt><dd> The implant material of embodiment 71, wherein said FCDP is provided in a sterile liquid. </dd></dl>

<dl><dt>75.</dt><dd> The implant material of embodiment 71, wherein said liquid is sodium acetate buffer. </dd></dl>

<dl><dt>76.</dt><dd> A method for preparing an implant material comprising soaking a material a calcium phosphate material in a sterile liquid comprising platelet-derived growth factor (FCDP) in a concentration in the range of about 0.1 mg / mL to about 1.0 mg / mL </dd></dl>

<dl><dt>77.</dt><dd> The method of embodiment 76, where the concentration of FCDP is approximately 0.3 mg / mL. </dd></dl>

<dl><dt>78.</dt><dd> The method of embodiment 76, wherein said calcium phosphate is selected from tricalcium phosphate, hydroxyapatite, low crystalline hydroxyapatite, amorphous calcium phosphate, calcium metaphosphate, dicalcium phosphate dihydrate, heptacalcium phosphate, calcium pyrophosphate dihydrate, calcium pyrophosphate and calcium phosphate.</dd></dl>