WO2003024316A2 - Agents porogenes pour ciments orthopediques - Google Patents

Agents porogenes pour ciments orthopediques Download PDF

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Publication number
WO2003024316A2
WO2003024316A2 PCT/US2002/029966 US0229966W WO03024316A2 WO 2003024316 A2 WO2003024316 A2 WO 2003024316A2 US 0229966 W US0229966 W US 0229966W WO 03024316 A2 WO03024316 A2 WO 03024316A2
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bmp
bone
pore
xaa
gdf
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PCT/US2002/029966
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English (en)
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WO2003024316A3 (fr
Inventor
Paresh S. Dalal
Tracy J. Landeryou
Carol Ann Toth
Shailesh C. Kulkarni
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Stryker Corporation
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Priority to JP2003528218A priority Critical patent/JP2005508217A/ja
Priority to AU2002327007A priority patent/AU2002327007B2/en
Priority to CA002460843A priority patent/CA2460843A1/fr
Priority to EP02761768A priority patent/EP1446445A4/fr
Publication of WO2003024316A2 publication Critical patent/WO2003024316A2/fr
Publication of WO2003024316A3 publication Critical patent/WO2003024316A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/02—Inorganic materials
    • A61L27/12—Phosphorus-containing materials, e.g. apatite
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/56—Porous materials, e.g. foams or sponges

Definitions

  • Bone tissue in the human body comprises the largest proportion of the body's connective tissue mass. However, unlike other connective tissues, its matrix consists of physiologically mineralized, tiny crystallites of a basic, carbonate-containing calcium phosphate called hydroxyapatite distributed in an organized collagen structure. Repair of this tissue is a complex process involving a number of complex cellular functions directed towards the formation of a scaffold and mineralization of the defect followed by an eventual remodeling of the defect site to attain the original structure. [0002] In a majority of situations, calcium phosphate based implants have been found to be compatible and conducive to bone repair.
  • Hydroxyapatite has a formula of Ca 10 (P0 4 ) 6 (OH) 2 and the compound is similar in stoichiometric composition to bone mineral and to tooth enamel. Porous hydroxyapatite blocks and particulates have been widely used as an implant to provide structural support as the material is osteoconductive and supports the ingrowth and attachment of bone. However, these hydroxyapatite materials have inconvenient handling properties for most orthopedic procedures (Parsons et al . , Annals of the New York Academy of Sciences, 523, pp. 190-207 (1988)).
  • Various calcium cement formulations i.e. hydroxyapatite cements
  • si tu Ti et al .
  • the present invention solves these problems by identifying a bone precursor composition comprising a cement mixture and a pore-forming agent.
  • the invention also provides a composition comprising a solid cement and a pore-forming agent.
  • the particle size of the pore-forming agent is 20-500 ⁇ m, more preferably 20- 140 ⁇ m, and most preferably, 75-140 ⁇ m.
  • the proportion of pore-forming agent is 10- 70% by volume, more preferably, 40-60% by volume.
  • the proportion of pore-forming is 7-40% by weight, more preferably, 7-25% by weight.
  • the proportion of PLGA is 7- 14% by weight.
  • the composition of this invention improves the penetration of cellular factors into the cement, while maintaining the physical strength and the handling properties of the implant, thereby improving the regeneration of bone tissue in a living body.
  • the invention also provides a composition comprising the bone precursor composition and a bioactive agent such as a bone morphogenic protein (BMP) or a nucleic acid molecule comprising a sequence encoding a BMP.
  • BMP bone morphogenic protein
  • the bioactive agent is encapsulated in the pore-forming agent.
  • the bone precursor composition or bone precursor composition/bioactive agent mixture can also be used in conjunction with binders to modulate the moldability of the composition at the implant site.
  • the invention also provides a kit comprising the bone precursor composition, and at least one or more additional components including a bioactive agent and a binder.
  • the invention also provides an implantable device comprising the bone precursor composition material, and optionally comprising one or more additional components including a bioactive agent such as a BMP or a binder.
  • the invention also provides an implantable prosthetic device comprising the bone precursor composition and optionally comprising one or more additional components including a bioactive agent such as a BMP or a binder.
  • the invention provides a method of inducing bone formation in a mammal comprising the step of implanting in the defect site a composition comprising the bone precursor composition and optionally a binder and/or a bioactive agent.
  • the invention also provides a method of delivering a bioactive agent at a site requiring bone formation comprising implanting at the defect site of a mammal a composition comprising the bone precursor composition and a bioactive agent .
  • Figure 1 Gross Image (top) and Faxitron Image (bottom) of right tibia of Animal 5334 at four weeks. From the left, the proximal site, middle site and distal site contain Formulations 1, 2 and 3, respectively. [0011] Figure 2. Gross Image (top) and Faxitron Image (bottom) of left tibia of Animal 5329 at four weeks. From the left, the proximal site, middle site and distal site contain Formulations 6, 7 and 8, respectively.
  • Figure 3 Gross Image (top) and Faxitron Image (bottom) of right tibia of Animal 5329 at four weeks. From the left, the proximal site, middle site and distal site contain Formulations Control, 10 and 9, respectively.
  • Figure 4 Gross Image (top) and Faxitron Image (bottom) of left tibia of Animal 5339 at eight weeks. From the left, the proximal site, middle site and distal site contain Formulations 3, 2 and 1, respectively. [0014] Figure 5. Gross Image (top) and Faxitron Image (bottom) of right tibia of Animal 5338 at eight weeks. From the left, the proximal site, middle site and distal site contain Formulations 6, 7 and 8, respectively. [0015] Figure 6. Gross Image (top) and Faxitron Image (bottom) of right tibia of Animal 5340 at eight weeks. From the left, the proximal site, middle site and distal site contain Formulations Control, 10 and 9, respectively.
  • amino acid sequence homology is understood to include both amino acid sequence identity and similarity. Homologous sequences share identical and/or similar amino acid residues, where similar residues are conservative substitutions for, or "allowed point mutations" of, corresponding amino acid residues in an aligned reference sequence. Thus, a candidate polypeptide sequence that shares 70% amino acid homology with a reference sequence is one in which any 70% of the aligned residues are either identical to, or are conservative substitutions of, the corresponding residues in a reference sequence. Certain particularly preferred morphogenic polypeptides share at least 60%, and preferably 70% amino acid sequence identity with the C-terminal 102-106 amino acids, defining the conserved seven-cysteine domain of human OP-1, BMP-2, and related proteins.
  • Amino acid sequence homology can be determined by methods well known in the art. For instance, to determine the percent homology of a candidate amino acid sequence to the sequence of the seven-cysteine domain, the two sequences are first aligned. The alignment can be made with, e . g. , the dynamic programming algorithm described in Needleman et al . , J. Mol . Biol . , 48, pp. 443 (1970) , and the Align Program, a commercial software package produced by DNAstar, Inc. The teachings of these references are incorporated herein by reference. An initial alignment can be refined by comparison to a multi-sequence alignment of a family of related proteins. Once the alignment is made and refined, a percent homology score is calculated.
  • the aligned amino acid residues of the two sequences are compared sequentially for their similarity to each other. Similarity factors include similar size, shape and electrical charge.
  • Similarity factors include similar size, shape and electrical charge.
  • One particularly preferred method of determining amino acid similarities is the PAM250 matrix described in Dayhoff et al . , Atlas of Protein Sequence and Structure, 5, pp. 345-352 (1978 & Supp . ) , which is incorporated herein by reference.
  • a similarity score is first calculated as the sum of the aligned pairwise amino acid similarity scores. Insertions and deletions are ignored for the purposes of percent homology and identity. Accordingly, gap penalties are not used in this calculation.
  • the raw score is then normalized by dividing it by the geometric mean of the scores of the candidate sequence and the seven-cysteine domain.
  • Bone tissue refers to a calcified (mineralized) connective tissue primarily comprising a composite of deposited calcium and phosphate in the form of hydroxyapatite, collagen (primarily Type I collagen) and bone cells such as osteoblasts, osteocytes and osteoclasts, as well as bone marrow tissue which forms in the interior of true endochondral bone. Bone tissue differs significantly from other tissues, including cartilage tissue.
  • bone tissue is vascularized tissue composed of cells and a biphasic medium comprising a mineralized, inorganic component (primarily hydroxyapatite crystals) and an organic component (primarily of Type I collagen) .
  • Glycosaminoglycans constitute less than 2% of this organic component and less than 1% of the biphasic medium itself, or of bone tissue per se .
  • the collagen present in bone tissue exists in a highly-organized parallel arrangement. Bony defects, whether from degenerative, traumatic or cancerous etiologies, pose a daunting challenge to the reconstructive surgeon. Particularly difficult is reconstruction or repair of skeletal parts that comprise part of a multi-tissue complex, such as occurs in mammalian joints.
  • Bone formation refers to formation of endochondral bone or formation of intramembranous bone. In humans, bone formation begins during the first 6-8 weeks of fetal development. Progenitor stem cells of mesenchymal origin migrate to predetermined sites, where they either: (a) condense, proliferate, and differentiate into bone-forming cells (osteoblasts) , a process observed in the skull and referred to as “intramembranous bone formation” or, (b) condense, proliferate and differentiate into cartilage-forming cells (chondroblasts) as intermediates, which are subsequently replaced with bone-forming cells. More specifically, mesenchymal stem cells differentiate into chondrocytes .
  • chondrocytes then become calcified, undergo hypertrophy and are replaced by newly formed bone made by differentiated osteoblasts, which now are present at the site. Subsequently, the mineralized bone is extensively remodeled, thereafter becoming occupied by an ossicle filled with functional bone-marrow elements. This process is observed in long bones and referred to as "endochondral bone formation".
  • bone In postfetal life, bone has the capacity to repair itself upon injury by mimicking the cellular process of embryonic endochondral bone development. That is, mesenchymal progenitor stem cells from the bone marrow, periosteum, and muscle can be induced to migrate to the defect site and begin the cascade of events described above. There, they accumulate, proliferate, and differentiate into cartilage, which is subsequently replaced with newly formed bone.
  • BMP Bone morphogenic protein
  • a protein belongs to the BMP family according to this invention when it has at least 50% amino acid sequence identity with at least one known BMP family member within the conserved C-terminal cysteine-rich domain which characterizes the BMP protein family.
  • Members of the BMP family may have less than 50% DNA or amino acid sequence identity overall.
  • Bone precursor composition refers to a composition comprising a cement mixture and a pore- forming agent.
  • the bone precursor composition is biocompatible and bioabsorbable.
  • the composition can be used to form a cement matrix for bone implant to form, repair or replace damaged connective tissue such as bone tissue.
  • Binder refers to any biocompatible material which, when admixed with the bone precursor composition and/or osteogenic protein, provides desired handling properties without adversely affecting bone formation. As taught herein, the skilled artisan can determine an effective amount of protein for use with any suitable binder using only routine experimentation. Among the other characteristics of. a preferred binder is an ability to render the device: pliable, shapeable and/or malleable; injectable; adherent to bone, cartilage, muscle and other tissues, resistant to disintegration upon washing and/or irrigating during surgery; and, resistant to dislodging during surgery, suturing and post-operatively, to name but a few.
  • Biocompatible refers to a material that does not elicit detrimental effects associated with the body's various protective systems, such as cell and humoral- associated immune responses, e.g., inflammatory responses and foreign body fibrotic responses.
  • the term biocompatible also implies that no specific undesirable cytotoxic or systemic effects are caused by the material when it is implanted into the patient.
  • cement mixture refers to a mixture that is the precursor of solid cement.
  • the mixture can be in a dry powder or granular form.
  • a liquid initiator Upon mixing with a liquid initiator, the mixture forms a plastic paste.
  • the paste undergoes a chemical reaction and/or a crystal rearrangement and hardens with time into a cured solid cement as a result of the hydration reaction.
  • the liquid initiator can be a physiologically acceptable aqueous initiator, e.g., water, an aqueous buffer or an aqueous solution.
  • the cement mixture can be used as a joiner, or filler for the assembly of connective tissue surfaces (e.g., bone tissue), which are not in direct contact, and to bond bone tissue to metallic or synthetic prosthetic devices.
  • the cement mixture is a calcium cement mixture. More preferably, the mixture is a calcium phosphate cement mixture, calcium sulfate cement mixture such as calcium sulfate hemihydrate, or a combination thereof.
  • Calcium phosphate cement mixture refers to a cement precursor composition that comprises at least two calcium phosphate compounds selected from the group consisting of calcium phosphate, amorphous calcium phosphate, decarbonated amorphous calcium phosphate, beta-tricalcium phosphate, alpha-tricalcium phosphate, monocalcium phosphate, dicalcium phosphate, octacalcium phosphate, calcium metaphosphate, heptacalcium phosphate, calcium pyrophosphate.
  • the calcium phosphate cement mixture forms calcium phosphate cement upon hydration and hardening.
  • Calcium sulfate cement mixture refers to a cement precursor composition that comprises a form of calcium sulfate including but not limited to calcium sulfate, calcium sulfate hemihydrate (Plaster of Paris) and calcium sulfate dihydrate (gypsum) .
  • the calcium sulfate cement mixture forms calcium sulfate cement upon hydration and hardening.
  • cement matrix refers to a composition that forms after mixing the bone precursor composition of the invention with the liquid initiator.
  • the cement matrix may be in a moldable putty form ready for implant or in a hardened solid form already .implanted in vivo or in si tu .
  • the hardened solid form has a scaffolding structure on which infiltrating cells can attach, proliferate and participate in the morphogenic process culminating in bone formation.
  • the cement matrix may also contain one or more components selected from a binder and a bioactive agent such as BMP.
  • Constant substitutions refers to residues that are physically or functionally similar to the corresponding reference residues.
  • a conservative substitution and its reference residue have similar size, shape, electric charge, chemical properties including the ability to form covalent or hydrogen bonds, or the like.
  • Preferred conservative substitutions are those fulfilling the criteria defined for an accepted point mutation in Dayhoff et al . , supra .
  • Examples of conservative substitutions are substitutions within the following groups: (a) valine, glycine; (b) glycine, alanine; (c) valine, isoleucine, leucine; (d) aspartic acid, glutamic acid; (e) asparagine, glutamine; (f) serine, threonine; (g) lysine, arginine, methionme; and (h) phenylalanine, tyrosine.
  • “conservative variant” or “conservative variation” also includes the use of a substituting amino acid residue in place of an amino acid residue in a given parent amino acid sequence, where antibodies specific for the parent sequence are also specific for, i.e., "cross-react” or “immuno-react” with, the resulting substituted polypeptide sequence.
  • “Defect” or “defect site” refers to a site requiring bone, joint, cartilage or ligament repair, construction, fusion, regeneration or augmentation. The site may be an orthopedic structural disruption or abnormality, or a site where bone does not normally grow. The defect further can define an osteochondral defect, including a structural disruption of both the bone and overlying cartilage.
  • a defect can assume the configuration of a "void", which is understood to mean a three-dimensional defect such as, for example, a gap, cavity, hole or other substantial disruption in the structural integrity of a bone or joint.
  • a defect can be the result of accident, disease, surgical manipulation, and/or prosthetic failure.
  • the defect is a void having a volume incapable of endogenous or spontaneous repair.
  • Such defects in long bone are generally twice the diameter of the subject bone and are also called “critical size” defects.
  • critical size defects are approximately 1.0 cm, and incapable of spontaneous repair. See, for example, Schmitz et al .
  • the gap is approximately 1.5 cm and 2.0 cm, respectively.
  • the defect is a non- critical size segmental defect. Generally, these are capable of spontaneous repair.
  • the defect is an osteochondral defect, such as an osteochondral plug. Such a defect traverses the entirety of the overlying cartilage and enters, at least in part, the underlying bony structure.
  • a chondral or subchondral defect traverses the overlying cartilage, in part or in whole, respectively, but does not involve the underlying bone.
  • Other defects susceptible to repair using the instant invention include, but are not limited to, non-union fractures; bone cavities; tumor resection; fresh fractures (distracted or undistracted) ; cranial, maxillofacial and facial abnormalities, for example, in facial skeletal reconstruction, specifically, orbital floor reconstruction, augmentation of the alveolar ridge or sinus, periodontal defects and tooth extraction socket; cranioplasty, genioplasty, chin augmentation, palate reconstruction, and other large bony reconstructions; vertebroplasty, interbody fusions in the cervical, thoracic and lumbar spine and posteriolateral fusions in the thoracic and lumbar spine; in osteomyelitis for bone regeneration; appendicular fusion, ankle fusion, total hip, knee and joint fusions or arthroplasty; correcting tendon and/or ligamentous tissue defects such as,
  • Morphogenic protein refers to a protein having morphogenic activity.
  • a morphogenic protein of this invention comprises at least one polypeptide belonging to the BMP protein family.
  • Morphogenic proteins may be capable of inducing progenitor cells to proliferate and/or to initiate differentiation pathways that lead to cartilage, bone, tendon, ligament, neural or other types of tissue formation depending on local environmental cues, and thus morphogenic proteins may behave differently in different surroundings. For example, an osteogenic protein may induce bone tissue at one treatment site and neural tissue at a different treatment site.
  • Ostogenic protein refers to a morphogenic protein that is capable of inducing a progenitor cell to form cartilage and/or bone.
  • the bone may be intramembranous bone or endochondral bone .
  • Most osteogenic proteins are members of the BMP protein family and are thus also BMPs. As described elsewhere herein, the class of proteins is typified by human osteogenic protein (hOP-1) .
  • osteogenic proteins useful in the practice of the invention include osteogenically active forms of OP-1, OP-2, OP-3, COP-1, COP-3, COP-4, COP-5, COP-7, COP-16, BMP-2, BMP-3, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14, BMP-15, BMP-16, BMP-17, BMP-18, GDF-1, GDF-2, GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-10, GDF-11, GDF-12,
  • osteogenic protein includes any one of: OP-1, OP-2, OP- 3, BMP-2, BMP-4, BMP-5, BMP-6, BMP-9, and amino acid sequence variants and homologs thereof, including species homologs thereof.
  • Particularly preferred osteogenic proteins are those comprising an amino acid sequence having at least 70% homology with the C-terminal 102-106 amino acids, defining the conserved seven cysteine domain, of human OP-1, BMP-2, and related proteins.
  • Certain preferred embodiments of the instant invention comprise the osteogenic protein, OP-1.
  • the osteogenic proteins suitable for use with applicants' invention can be identified by means of routine experimentation using the art-recognized bioassay described by Reddi and Sampath
  • Proteins useful in this invention include eukaryotic proteins identified as osteogenic proteins
  • BMP-3 is also preferred. Additional useful proteins include biosynthetic morphogenic constructs disclosed in U.S. Pat. No. 5,011,691, incorporated herein by reference, e.g., COP-1, COP-3, COP-4, COP-5, COP-7 and COP-16, as well as other proteins known in the art. Still other proteins include osteogenically active forms of BMP-3b (see Takao, et al . Biochem. Biophys . Res. Comm.
  • BMP-9 see WO95/33830
  • BMP-15 see WO96/35710
  • BMP-12 see WO95/16035)
  • CDMP-1 see WO 94/12814)
  • CDMP-2 see W094/12814)
  • BMP-10 see W094/26893
  • GDF-1 see WO92/00382
  • GDF-10 see WO95/10539
  • GDF-3 see W094/15965
  • GDF-7 see WO95/01802 .
  • Solid cement refers to the cured, hardened solid as a result of the hydration reaction of the cement mixture. Depending on the composition of the cement mixture, it can take from a few minutes to a few hours for the cement mixture to form a solid cement.
  • the solid cement is preferably calcium phosphate cement or calcium sulfate cement.
  • the calcium phosphate cement can be in a form of hydroxyapatite such as hydroxyapatite, carbonated hydroxyapatite or poorly-crystalline hydroxyapatite.
  • the solid cement can be formed in vivo or in si tu to induce bone growth, repair or replace damaged connective tissue such as bone tissue.
  • the present invention provides a bone precursor composition comprising a cement mixture and a pore- forming agent.
  • Preferred cement mixtures are calcium cement mixtures. More preferably, the calcium cement mixture is a calcium phosphate cement mixture or a calcium sulfate cement mixture.
  • the cement mixture Upon hydration and setting at ambient temperature, e.g., room or body temperature, the cement mixture forms a solid cement. Hydration can be achieved by the addition of liquid such as water, saline buffer or an aqueous solution.
  • the setting time for these cements may range from a few minutes to a few hours depending on their composition and the amount of liquid added. Buffers such as sodium phosphate or sodium pyrophosphate can reduce the setting time.
  • the calcium phosphate cement mixture is selected from the group consisting of a mixture of beta-tricalcium phosphate ( ⁇ -TCP) and monocalcium phosphate monohydrate (MCPM) ; a mixture of ⁇ - TCP, dicalcium phosphate dihydrate (DCPD) and calcium carbonate (CC) ; a mixture of monocalcium phosphate, tricalcium phosphate and calcium carbonate; a mixture of a decarbonated amorphous calcium phosphate and .a second calcium phosphate; a mixture of tetracalcium phosphate (TTCP) and a second calcium phosphate.
  • ⁇ -TCP beta-tricalcium phosphate
  • MCPM monocalcium phosphate monohydrate
  • DCPD dicalcium phosphate dihydrate
  • CC calcium carbonate
  • TTCP tetracalcium phosphate
  • the second calcium phosphate is selected from the group consisting of monocalcium phosphate, dicalcium phosphate anhydrous, dicalcium phosphate dihydrate, calcium metaphosphate, heptacalcium phosphate, calcium pyrophosphate, alpha-tricalcium phosphate, beta-tricalcium phosphate, octacalcium phosphate and amorphous calcium phosphate.
  • the calcium phosphate cement mixture comprises a mixture of beta- tricalcium phosphate ( ⁇ -TCP) and monocalcium phosphate monohydrate (MCPM) . More preferably, the ⁇ -TCP/MCPM mixture further comprises calcium pyrophosphate (CPP) , calcium sulfate dihydrate (CSD) and calcium sulfate hemihydrate (CSH) .
  • CPP calcium pyrophosphate
  • CSD calcium sulfate dihydrate
  • CSH calcium sulfate hemihydrate
  • the calcium phosphate cement mixture comprises a mixture of ⁇ -TCP, dicalcium phosphate dihydrate (DCPD) and calcium carbonate (CC) .
  • the ⁇ -TCP/DCPD/CC mixture further comprises hydroxyapatite. In the presence of water, the mixture produces hydroxyapatite.
  • the setting time of the ⁇ -TCP, DCPD and CC mixture can be reduced from 4.5 hours to about 20 minutes by the addition of small amounts (8% (w/w) ) of hydroxyapatite.
  • the calcium phosphate cement mixture comprises a mixture of monocalcium phosphate, tricalcium phosphate and calcium carbonate.
  • the calcium phosphate cement mixture comprises a mixture of decarbonated amorphous calcium phosphate and a second calcium phosphate, wherein the second calcium phosphate is selected from the group consisting of dicalcium phosphate dihydrate, calcium metaphosphate, heptacalcium phosphate, calcium pyrophosphate and tricalcium phosphate.
  • the decarbonated amorphous calcium phosphate is formed by heating an amorphous carbonated calcium phosphate to remove a portion of the carbonate component.
  • the amorphous carbonated calcium phosphate is precipitated from an aqueous solution comprising calcium ions, phosphate ions and carbonate ions having a calcium to phosphorous ratio in the range of about 1.55 to 1.7.
  • aqueous solution comprising calcium ions, phosphate ions and carbonate ions having a calcium to phosphorous ratio in the range of about 1.55 to 1.7.
  • body temperature 37 °C
  • mixing the decarbonated amorphous calcium phosphate and a second calcium phosphate in water or buffer results in a poorly- crystalline hydroxyapatite which forms within 20 minutes.
  • Poorly-crystalline hydroxyapatite contains nanometer- sized crystals and has substantially the same X-ray diffraction spectrum as bone.
  • the poorly-crystalline cement is more soluble and provides better osteoconductive cell mediated absorption.
  • the solubility of the poorly- crystalline hydroxyapatite can be further improved by modifying the Ca/P ratio.
  • the poorly-crystalline cement is particularly suitable for the incorporation of bioactive agents, such as BMPs since the setting reaction produces minimal heat, which minimizes the denaturing of protein structure.
  • the calcium phosphate cement mixture is tetracalcium phosphate (TTCP) and a second calcium phosphate; wherein the second calcium phosphate is selected from the group consisting of monocalcium phosphate, dicalcium phosphate anhydrous (DCPA) , dicalcium phosphate dihydrate (DCPD) , alpha-tricalcium phosphate, beta-tricalcium phosphate, octacalcium phosphate, and amorphous calcium phosphate.
  • DCPA dicalcium phosphate anhydrous
  • DCPD dicalcium phosphate dihydrate
  • alpha-tricalcium phosphate beta-tricalcium phosphate
  • octacalcium phosphate octacalcium phosphate
  • amorphous calcium phosphate amorphous calcium phosphate
  • the tetracalcium phosphate has a molar Ca/P ratio below 2:1. If the ratio is above 2:1, calcium oxide may be present as an impurity. This causes the pH of the cement slurry to rise substantially above pH 8.5, which impedes the setting reaction. It is critical to maintain the tetracalcium phosphate under anhydrous conditions. If not, the compound will be less reactive in forming cement.
  • the second calcium phosphate is selected from the group consisting of dicalcium phosphate anhydrous (DCPA) and dicalcium phosphate dihydrate (DCPD) . Relatively large TTCP and small DCPA particles help to achieve rapid setting and high strength in the cement.
  • the TTCP/DCPA ratio can range from 1:1 to 1:4. Preferably, the ratio is 1:1. A 1:1 ratio produces hydroxyapatite.
  • the calcium sulfate cement mixture used in the invention comprises calcium sulfate hemihydrate (CSH, CaS0 4 .1/2H 2 0) .
  • CSH is produced by heating gypsum (CaSO 4 .2H 2 0) so that it loses 75% of its water.
  • gypsum CaSO 4 .2H 2 0
  • the invention also provides a composition comprising a solid cement formed from the cement mixture and a pore-forming agent.
  • the solid cement is a calcium phosphate cement or a calcium sulfate cement .
  • Calcium phosphate cements include but are not limited to hydroxyapatite, poorly-crystalline HA cement or carbonated hydroxyapatite.
  • the pore-forming agents of this invention may be in bead or resin form.
  • the pore-forming agents can be resorbable biocompatible polymers including both natural and synthetic polymers. Natural polymers are typically absorbed by enzymatic degradation in the body, while synthetic resorbable polymers typically degrade by a hydrolytic mechanism.
  • the pore-forming agent is selected from the group consisting of ethylenevinylacetate, natural and synthetic collagen, pol (glaxanone) , poly (phosphazenes) , polyglactin, polyglactic acid, polyaldonic acid, polyacrylic acids, polyalkanoates, polyorthoesters, poly (L-lactide) (PLLA) , poly (D, L-lactide) (PDLLA) , polyglycolide (PGA), poly (lactide-co-glycolide (PLGA) , poly ( ⁇ -caprolactone) , poly (trimethylene carbonate) , poly (p-dioxanone) , poly ( ⁇ -caprolactone-co-glycolide) , poly (glycolide-co-trimethylene carbonate) poly (D,L-lactide-co-trimethylene carbonate), polyarylates, polyhydroxybutyrate (PHB) , polyanhydrides, poly
  • the pore- forming agent is selected from the group consisting of polyorthoesters, poly (L-lactide) (PLLA) , poly (D, L-lactide) (PDLLA) , polyglycolide (PGA), poly (lactide-co-glycolide (PLGA) , poly ( ⁇ -caprolactone) , poly (trimethylene carbonate), poly (p-dioxanone) , poly ( ⁇ -caprolactone-co-glycolide) , poly (glycolide-co-trimethylene carbonate) , poly (D, L-lactide-co-trimethylene carbonate), polyarylates and co-polymers thereof.
  • polyorthoesters poly (L-lactide) (PLLA) , poly (D, L-lactide) (PDLLA) , polyglycolide (PGA), poly (lactide-co-glycolide (PLGA) , poly ( ⁇ -caprolactone)
  • the pore- forming agent is selected from the group consisting of ethylenevinylacetate, natural and synthetic collagen, poly (glaxanone) , poly (phosphazenes) , polyglactin, polyglactic acid, polyaldonic acid, polyacrylic acids, polyalkanoates and co-polymers thereof.
  • the pore-forming agent is selected from the group consisting of polyhydroxybutyrate (PHB) , anhydrides including polyanhydrides, poly (anhydride-co-imide) and co-polymers thereof, polymers of amino acids, propylene-co-fumarates, a polymer of one or more -hydroxy carboxylic acid monomers, (e.g., -hydroxy acetic acid (glycolic acid) and/or ⁇ -hydroxy propionic acid (lactic acid) ) , calcium sulfate and bioactive glass compositions.
  • ⁇ -hydroxy propionic acid can be employed in its d- or 1- form, or as a racemic mixture.
  • the pore- forming agent is selected from the group consisting of poly (lactide-co-glycolide) (PLGA) and calcium sulfate.
  • PLGA poly (lactide-co-glycolide)
  • the molar ratio of the lactide, glycolide monomers can be adjusted. In a preferred embodiment, the monomer ratio is 50:50.
  • the molecular weight range of the polymer is from about 5,000 to 100,000 daltons, more preferably 10,000 to 30,000 daltons.
  • PLGA has a more rapid resorption rate than calcium sulfate.
  • the degradation of PLGA is through chemical hydrolysis of the hydrolytically unstable backbone .
  • the calcium phosphate cement mixture comprises a mixture of tetracalcium phosphate and dicalcium phosphate anhydrous; and the pore-forming agent is selected from the group consisting of PLGA and calcium sulfate.
  • the calcium sulfate cement mixture comprises calcium sulfate hemihydrate; and the pore- forming agent is PLGA.
  • the resorption rate of the pore-forming agent is faster than that of the nonresorbable calcium cement. As a result, pores are formed in the calcium cement.
  • the rate of resorption of the pore-forming agent is dependent on the proportion, polymer type and particle size of the pore-forming agent.
  • the mechanical strength of the implant decreases as the proportion of the pore-forming agent increases. An excessive amount of pore-forming agent leads to a decrease in the density of the cement body which results in lower mechanical strength. A deficiency in the amount of pore-forming agent may result in insufficient pores in the cement.
  • the proportion of pore-forming agent is 10- 70% by volume, more preferably, 40-60% by volume.
  • the proportion of pore-forming agent is preferably 7-40% by weight, more preferably 7-25% by weight, most preferably 7-14% by weight when the pore-forming agent is PLGA.
  • the particle size of the pore-forming agent will influence the pore size generated in the cement. A sufficient pore size is required to provide residence spaces for the infiltrating osteolytic cells and osteoblasts. The pore size also controls the rate of resorption. Bone repair is optimal when the rate of resorption coincides with the rate of bone growth. It is preferred that the pore-forming agent creates a pore size diameter of 20-500 ⁇ m, preferably 20-140 ⁇ m, more preferably 50-140 ⁇ m, and most preferably 75-140 ⁇ m.
  • the bone precursor composition of this invention may also be combined with one or more bioactive agents.
  • the bioactive agent may be an agent that enhances bone growth.
  • the bioactive agent is a bone morphogenic protein.
  • the bone morphogenic protein is selected from the group consisting of OP-1 (BMP-7) , OP-2, OP-3, COP-1, COP-3, COP-4, COP-5, COP-7, COP-16, BMP-2, BMP-3, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14, BMP-15, BMP-16, BMP-17, BMP-18, GDF-1, GDF-2, GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-10, GDF-11, GDF-12, MP121, dorsalin-1, DPP, Vg-1, Vgr-1, 60A protein, NODAL, UNIVIN, SCREW, ADMP, NEURAL, and TGF- ⁇ .
  • the morphogenic protein is OP-1.
  • the bioactive agent is encapsulated in the pore-forming agent. As the pore-forming agent is slowly resorbed by the osteoclast cells, the encapsulated bioactive agent is gradually released into the matrix.
  • the biodegradable agent is multi-layered. Each layer comprises a different biodegradable agent, preferably, differing in the rate of resorption.
  • Methods of encapsulating the bioactive agent include but are not limited to the emulsion-solvent evaporation method (Grandfils et al . , Journal of Biomedical Materials Research, 26, pp. 467-479 (1992)) and the method described in Herbert et al . , Pharmaceutical Research, 15, pp. 357-361 (1998) .
  • the above two references are incorporated herein by reference.
  • the latter method is especially suitable for encapsulating proteins.
  • Other methods are described in U.S. patents 6,110,503, 5,654,008 and 5,271,961, which are incorporated herein by reference.
  • the bioactive agent is stabilized by the addition of lactose during the encapsulation process.
  • the bioactive agent is a repair cell.
  • the repair cell is a mammalian cell, more preferably, a human cell of the same type as that of the tissue being repaired or reconstructed. Suitable examples of repair cells include bone cells such as bone marrow stem cells, osteocytes, osteoblasts, osteoclasts and bone progenitor cells.
  • the cell is transfected with a nucleic acid molecule encoding a BMP.
  • the bioactive agent is a nucleic acid molecule comprising a sequence encoding a BMP, preferably, OP-1 (SEQ ID NO: 10) .
  • the nucleic acid molecule is a RNA or DNA molecule.
  • the nucleic acid sequence encoding the BMP may be inserted in recombinant expression vectors. Examples of vectors include but are not limited to pBR322, pH717, pH731, pH752, pH754 and pW24. SP6 vectors may be used for in vi tro transcription of RNA.
  • Transcription promoters useful for expressing the BMP include but are not limited to the SV40 early promoter, the adenovirus promoter (AdMLP) , the mouse metallothionein-I promoter (mMT-I) , the Rous sarcoma virus (RSV) long terminal repeat (LTR) , the mouse mammary tumor virus long terminal repeat (MMTV-LTR) , and the human cytomegalovirus major intermediate-early promoter (hCMV) .
  • AdMLP adenovirus promoter
  • mMT-I mouse metallothionein-I promoter
  • RSV Rous sarcoma virus
  • LTR Rous sarcoma virus
  • MMTV-LTR mouse mammary tumor virus long terminal repeat
  • hCMV human cytomegalovirus major intermediate-early promoter
  • the DNA sequence may also be inserted in the genome of a recombinant virus such as, for example recombinant adenovirus, adeno-associated virus or retrovirus.
  • the repair cell or bone progenitor cell is then transfected or infected with the vector or virus to express the BMP protein.
  • the nucleic acid sequence may transiently or stably transfect the repair cell or bone progenitor cell.
  • the nucleic acid molecule is directly injected into the implant site.
  • the nucleic acid molecule is encapsulated in the pore-forming agent, preferably, in PLGA of 25-30 kD.
  • the nucleic acid molecule is trapped in a carrier selected from the group consisting of mannitol, sucrose, lactose, trehalose, liposomes, proteoliposomes that contain viral envelope proteins and polylysine- glycoprotein complexes. See, e . g. , Ledley, J. Pediatrics 110, pp. 1 (1987); Nicolau et al . , Proc . Natl. Acad. Sci . U.S.A. , 80, pp. 1068 (1983) .
  • the nucleic acid is transfected or infected into target cells such as bone progenitor cells and repair cells that have been removed from the body. The transfected cells are then re-implanted into the body.
  • the invention also provides a method of producing the bone precursor composition.
  • the cement mixture in dried powdered form is blended with the pore- forming agent to form an evenly distributed mixture of the bone precursor composition. This allows the cement mixture to surround the pore-forming agents so that pores are formed in the cement matrix in si tu .
  • Water, buffer or an aqueous solution is then added to the composition.
  • the paste is allowed to set and harden to form a composition comprising a pore-forming agent dispersed in a solid cement.
  • This method is distinguishable from methods of preparing formulations wherein the cement mixture is added to a biodegradable material in liquid form. There, the biodegradable material serves as a matrix around the cement instead of a pore-forming agent.
  • the bone precursor composition of this invention may further be combined with a biocompatible binder.
  • the binder provides enhanced viscosity and cohesiveness of the composition, allowing the skilled practitioner to position and shape the composition within the voids, defects or other areas in which new bone growth is desired.
  • the enhanced cohesiveness of the composition also prevents particle migration associated with grafting materials for orthopedic, maxillofacial and dental applications.
  • the minimum amount of binder is that amount required to give easy formability and provide sufficient particle cohesion and shape retention during the period of tissue ingrowth.
  • the binder according to this invention is biodegradable, biocompatible and has fluid flow properties.
  • the binders contemplated as useful herein include, but are not limited to art-recognized suspending agents, viscosity-producing agents, gel-forming agents and emulsifying agents.
  • Other binders include agents used to suspend ingredients for topical, oral or parental administration.
  • Yet other binders are agents useful as tablet binders, disintegrants or emulsion stabilizers.
  • Still other binders are agents used in cosmetics, toiletries and food products (See USP XXII -NF XVII The Nineteen Ninety U.S. Pharmacopeia and the National Formulary (1990) ) .
  • Other components including antioxidants such as EDTA, citrate, butylated hydroxytoluene (BHT) , and surfactants such as poly (sorbates) and poly (oxyethylenes) may be added to the binder.
  • a preferred binder is carboxymethylcellulose (CMC) sodium.
  • Carboxymethylcellulose sodium is the sodium salt of a polycarboxymethyl ether of cellulose with a typical molecular weight ranging from 90,000 -
  • binders also include reagents such as gelatin, that are solubilized in warm or hot aqueous solutions, and are transformed into a non-flowable gel upon cooling.
  • the gelatin composition is formulated so that the composition is flowable at temperatures above body temperature of the mammal for implant, but transitions to relatively non-flowable gel at or slightly above such body temperature .
  • the binder is selected from the group consisting of sodium alginate, hyaluronic acid, sodium hyaluronate, gelatin, peptides, mucin, chrondroitin sulfate, chitosan, poloxamer, glycosaminoglycan, polysaccharide, polyethylene glycol, methylcellulose, carboxy methylcellulose, carboxy methylcellulose sodium, carboxy methylcellulose calcium, hydroxypropyl methylcellulose, hydroxybutyl methylcellulose, hydroxyethyl methylcellulose, hydroxyethylcellulose, methylhydroxyethyl cellulose, hydroxyethyl cellulose, mannitol, white petrolatum, mannitol/dextran combinations, mannitol/white petrolatum combinations, sesame oil, fibrin glue, blood and admixtures thereof .
  • the binder is selected from the group consisting of sodium alginate, hyaluronic acid, methylcellulose, carboxy methylcellulose, carboxy methylcellulose sodium, carboxy methylcellulose calcium, hydroxypropyl methylcellulose, hydroxybutyl methylcellulose, hydroxyethyl methylcellulose, hydroxyethylcellulose, methylhydroxyethyl cellulose, hydroxyethyl cellulose and admixtures thereof.
  • the binder is selected from the group consisting of sodium alginate, hyaluronic acid, carboxy methylcellulose, carboxy methylcellulose sodium and carboxy methylcellulose calcium.
  • the binder of this invention is selected from a class of high molecular weight hydrogels including sodium hyaluronate ( ⁇ 500-3000 kD) , chitosan ( ⁇ 100 - 300 kD) , poloxamer (- 7 - 18 kD) , and glycosaminoglycan ( ⁇ 2000 - 3000 kD) .
  • the glycosaminoglycan is N, O-carboxymethylchitosan glucosamine.
  • Hydrogels are cross-linked hydrophilic polymers in the form of a gel which have a three-dimensional network. Hydrogel may carry a net positive charge, a net negative charge, or may be neutral .
  • a typical net negative charged hydrogel is alginate.
  • Hydrogels carrying a net positive charge may be typified by extracellular matrix components such as collagen and laminin.
  • extracellular matrix components such as collagen and laminin.
  • extracellular matrix components include MatrigelTM (Dulbecco ' s modified eagle's medium with 50 ⁇ g/ml gentamicin) and VitrogenTM (a sterile solution of purified, pepsin-solubilized bovine dermal collagen dissolved in 0.012 N HCl) .
  • Examples of a net neutral hydrogel is highly crosslinked polyethylene oxide and polyvinyalcohol .
  • the binder is polyethylene glycol .
  • a mixture of low- and high- molecular-weight polyethylene glycols produce a paste with the proper viscosity.
  • a mixture of polyethylene glycols of molecular weight 400-600 daltons and 1500 daltons at the proper ratio are effective in forming a binder according to this invention.
  • the binder is selected from a class of polysaccharides with an average molecular weight of about 200,000 to 5,000,000 daltons consisting of dextran, dextran sulfate, diethylaminoethyl dextran, dextran phosphate or mixtures thereof.
  • Lower molecular weight polysaccharides have a faster dextran absorption rate, which results in earlier exposure of the porous bone precursor composition material. If it is desired that dextrans remain in the site for an extended period, dextrans of relatively high molecular weight may be used.
  • Other preferred polysaccharides include starch, fractionated starch, amylopectin, agar, gum arable, pullullan, agarose, carrageenan, dextrins, fructans, inulin, mannans, xylans, arabinans, glycogens, glucans, xanthan gum, guar gum, locust bean gum, tragacanth gum, karaya gum, and derivatives and mixtures thereof.
  • the binder is selected from the group consisting of mannitol , white petrolatum, mannitol/dextran combinations, mannitol/white petrolatum combinations, sesame oil, fibrin glue and admixtures thereof.
  • the binder is fibrin glue. Fibrin glue comprises a mixture of mammalian fibrinogen and thrombin.
  • Fibrin glue may also be made of fibrinogen and thrombin from other mammalian sources, such as, for example, bovine and murine sources.
  • the binder is human blood, preferably autogenous blood. This kind of binder serves as a protein-sequestering material.
  • the invention also relates to a kit for bone implant comprising the bone precursor composition material of the invention and a bioactive agent such as bone morphogenic protein.
  • the kit further comprises a binder.
  • the kit comprises the bone precursor composition material of the invention and a binder.
  • the BMP family named for its representative bone morphogenic/osteogenic protein family members, belongs to the TGF- ⁇ protein superfamily. Of the reported "BMPs" (BMP-1 to BMP-18) , isolated primarily based on sequence homology, all but BMP-1 remain classified as members of the BMP family of morphogenic proteins (Ozkaynak et al . , EMBO J. , 9, pp. 2085-93 (1990) ) .
  • the BMP family includes other structurally-related members which are morphogenic proteins, including the drosophila decapentaplegic gene complex (DPP) products, the Vgl product of Xenopus laevis and its murine homolog, Vgr-1 (see, e . g. , Massague, Annu . Rev . Cell Biol. , 6, pp. 597-641 (1990), incorporated herein by reference) .
  • DPP drosophila decapentaplegic gene complex
  • Vgr-1 see, e . g. , Massague, Annu . Rev . Cell Biol. , 6, pp. 597-641 (1990), incorporated herein by reference
  • the C-terminal domains of BMP-3, BMP-5, BMP-6, and OP-1 (BMP-7) are about 60% identical to that of BMP- 2, and the C-terminal domains of BMP-6 and OP-1 are 87% identical.
  • BMP-6 is likely the human homolog of the murine Vgr-1 (Lyons et al . , Proc . Natl . Acad. Sci . U.S.A. , 86, pp. 4554-59 (1989)); the two proteins are 92% identical overall at the amino acid sequence level (U. S. Patent No. 5,459,047, incorporated herein by reference) . BMP-6 is 58% identical to the Xenopus Vg-1 product.
  • the naturally occurring bone morphogens share substantial amino acid sequence homology in their C- terminal regions (domains) .
  • the above- mentioned naturally occurring osteogenic proteins are translated as a precursor, having an N-terminal signal peptide sequence typically less than about 30 residues, followed by a "pro" domain that is cleaved to yield the mature C-terminal domain of approximately 100-140 amino acids.
  • the signal peptide is cleaved rapidly upon translation, at a cleavage site that can be predicted in a given sequence using the method of Von Heijne Nucleic Acids Research, 14, pp. 4683-4691 (1986) .
  • the pro domain typically is about three times larger than the fully processed mature C-terminal domain.
  • BMP protein family members Another characteristic of the BMP protein family members is their apparent ability to dimerize.
  • OPs bone-derived osteogenic proteins
  • BMPs bone-derived osteogenic proteins
  • the ability of OPs and BMPs to form heterodimers may confer additional or altered morphogenic inductive capabilities on morphogenic proteins.
  • Heterodimers may exhibit qualitatively or quantitatively different binding affinities than homodimers for OP and BMP receptor molecules. Altered binding affinities may in turn lead to differential activation of receptors that mediate different signaling pathways, which may ultimately lead to different biological activities or outcomes.
  • the pair of morphogenic polypeptides have amino acid sequences each comprising a sequence that shares a defined relationship with an amino acid sequence of a reference morphogen.
  • preferred osteogenic polypeptides share a defined relationship with a sequence present in osteogenically active human OP-1, SEQ ID NO: 1.
  • any one or more of the naturally occurring or biosynthetic sequences disclosed herein similarly could be used as a reference sequence.
  • Preferred osteogenic polypeptides share a defined relationship with at least the C-terminal six cysteine domain of human OP-1, residues 335-431 of SEQ ID NO: 1.
  • osteogenic polypeptides share a defined relationship with at least the C-terminal seven cysteine domain of human OP-1, residues 330-431 of SEQ ID NO: 1. That is, preferred polypeptides in a dimeric protein with bone morphogenic activity each comprise a sequence that corresponds to a reference sequence or is functionally equivalent thereto.
  • Functionally equivalent sequences include functionally equivalent arrangements of cysteine residues disposed within the reference sequence, including amino acid insertions or deletions which alter the linear arrangement of these cysteines, but do not materially impair their relationship in the folded structure of the dimeric morphogen protein, including their ability to form such intra- or inter-chain disulfide bonds as may be necessary for morphogenic activity.
  • Functionally equivalent sequences further include those wherein one or more amino acid residues differs from the corresponding residue of a reference sequence, e . g. , the C-terminal seven cysteine domain (also referred to herein as the conserved seven cysteine skeleton) of human OP-1, provided that this difference does not destroy bone morphogenic activity.
  • Natural-sourced osteogenic protein in its mature, native form is a glycosylated dimer typically having an apparent molecular weight of about 30-36 kDa as determined by SDS-PAGE. When reduced, the 30 kDa protein gives rise to two glycosylated peptide subunits having apparent molecular weights of about 16 kDa and 18 kDa.
  • the unglycosylated protein which also has osteogenic activity, has an apparent molecular weight of about 27 kDa. When reduced, the 27 kDa protein gives rise to two unglycosylated polypeptides, having molecular weights of about 14 kDa to
  • Osteogenic proteins may include forms having varying glycosylation patterns, varying N-termini, and active truncated or mutated forms of native protein.
  • particularly useful sequences include those comprising the C-terminal 96 or 102 amino acid sequences of DPP (from Drosophila) , Vgl (from Xenopus) , Vgr-1 (from mouse), the OP-1 and OP-2 proteins, (see U.S. Pat. No. 5,011,691 and Oppermann et al .
  • BMP-2 proteins referred to as BMP-2, BMP-3, BMP-4
  • BMP-5 proteins referred to as BMP-5
  • BMP-6 proteins referred to as BMP-8
  • BMP-9 proteins referred to as BMP-8, BMP-9.
  • Preferred morphogenic and osteogenic proteins of this invention comprise at least one polypeptide selected from the group consisting of OP-1 (BMP-7), OP-2, OP-3, COP-1, COP-3, COP-4, COP-5, COP-7, COP-16, BMP-2, BMP-3, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14, BMP-15, BMP-16, BMP-17, BMP-18, GDF-1, GDF-2, GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-10, GDF-11, GDF-12, MP121, dorsalin-1, DPP, Vg-1, Vgr-1, 60A protein, NODAL, UNIVIN, SCREW, ADMP, NEURAL, TGF- ⁇ and amino acid sequence variants and homologs thereof, including species homologs, thereof.
  • BMP-7 OP-1
  • the morphogenic protein comprises at least one polypeptide selected from the group consisting of OP-1 (BMP-7), BMP-2, BMP-4, BMP-5 and BMP-6; more preferably, OP-1 (BMP-7) and BMP-2; and most preferably, OP-1 (BMP-7) .
  • BMP-9 WO95/33830 (PCT/US95/07084)
  • BMP-10 W094/26893 (PCT/US94/05290)
  • BMP-11 W094/26892
  • GDF-8 W094/21681 (PCT/US94/03019)
  • GDF-9 W094/15966 (PCT/US94/00685)
  • GDF-10 WO95/10539
  • useful proteins include biologically active biosynthetic constructs, including novel biosynthetic morphogenic proteins and chimeric proteins designed using sequences from two or more known morphogens .
  • a morphogenic protein may be prepared synthetically to induce tissue formation.
  • Morphogenic proteins prepared synthetically may be native, or may be non-native proteins, i.e., those not otherwise found in nature.
  • Non-native osteogenic proteins have been synthesized using a series of consensus DNA sequences (U.S. Patent No. 5,324,819, incorporated herein by reference). These consensus sequences were designed based on partial amino acid sequence data obtained from natural osteogenic products and on their observed homologies with other genes reported in the literature having a presumed or demonstrated developmental function. [0083] Several of the biosynthetic consensus sequences (called consensus osteogenic proteins or "COPs”) have been expressed as fusion proteins in prokaryotes. Purified fusion proteins may be cleaved, refolded, implanted in an established animal model and shown to have bone- and/or cartilage-inducing activity.
  • COPs biosynthetic consensus sequences
  • the currently preferred synthetic osteogenic proteins comprise two synthetic amino acid sequences designated COP-5 (SEQ. ID NO: 2) and COP-7 (SEQ. ID NO: 3) [0084] Oppermann et al . , U. S. Patent Nos. 5,011,691 and 5,324,819, which are incorporated herein by reference, describe the amino acid sequences of COP-5 and COP-7 as shown below:
  • the DNA and amino acid sequences of these and other BMP family members are published and may be used by those of skill in the art to determine whether a newly identified protein belongs to the BMP family.
  • New BMP- related gene products are expected by analogy to possess at least one morphogenic activity and thus classified as a BMP.
  • the morphogenic protein comprises a pair of subunits disulfide bonded to produce a dimeric species, wherein at least one of the subunits comprises a recombinant peptide belonging to the BMP protein family.
  • the morphogenic protein comprises a pair of subunits that produce a dimeric species formed through non-covalent interactions, wherein at least one of the subunits comprises a recombinant peptide belonging to the BMP protein family.
  • Non-covalent interactions include Van der Waals, hydrogen bond, hydrophobic and electrostatic interactions.
  • the dimeric species may be a homodimer or heterodimer and is capable of inducing cell proliferation and/or tissue formation.
  • bone morphogenic proteins useful herein include those in which the amino acid sequences comprise a sequence sharing at least 70% amino acid sequence homology or "similarity", and preferably 80% homology or similarity, with a reference morphogenic protein selected from the foregoing naturally occurring proteins.
  • the reference protein is human OP-1, and the reference sequence thereof is the C-terminal seven cysteine domain present in osteogenically active forms of human OP-1, residues 330- 431 of SEQ ID NO : 1.
  • a polypeptide suspected of being functionally equivalent to a reference morphogen polypeptide is aligned therewith using the method of Needleman, et al .
  • Bone morphogenic proteins useful herein accordingly include allelic, phylogenetic counterpart and other variants of the preferred reference sequence, whether naturally-occurring or biosynthetically produced (e. g. , including "muteins” or “mutant proteins”) , as well as novel members of the general morphogenic family of proteins, including those set forth and identified above.
  • osteogenic proteins include those sharing the conserved seven cysteine domain and sharing at least 70% amino acid sequence homology (similarity) within the C-terminal active domain, as defined herein.
  • the osteogenic proteins of the invention can be defined as osteogenically active proteins having any one of the generic sequences defined herein, including OPX (SEQ ID NO: 4) and Generic Sequences 7 (SEQ ID NO: 5) and 8 (SEQ ID NO: 6), or Generic Sequences 9 (SEQ ID NO: 7) and 10 (SEQ ID NO: 8) .
  • the family of bone morphogenic polypeptides useful in the present invention, and members thereof, can be defined by a generic amino acid sequence.
  • Generic Sequence 7 (SEQ ID NO : 5) and Generic Sequence 8 (SEQ ID NO: 6) are 97 and 102 amino acid sequences, respectively, and accommodate the homologies shared among preferred protein family members identified to date, including at least OP-1, OP-2, OP-3, CBMP-2A, CBMP-2B, BMP-3, 60A, DPP, Vgl, BMP-5, BMP-6, Vgr-1, and GDF-1.
  • the amino acid sequences for these proteins are described herein and/or in the art, as summarized above.
  • the generic sequences include both the amino acid identity shared by these sequences in the C-terminal domain, defined by the six and seven cysteine skeletons (Generic Sequences 7 and 8, respectively), as well as alternative residues for the variable positions within the sequence.
  • the generic sequences provide an appropriate cysteine skeleton where inter- or intramolecular disulfide bonds can form, and contain certain critical amino acids likely to influence the tertiary structure of the folded proteins.
  • the generic sequences allow for an additional cysteine at position 36 (Generic Sequence 7) or position 41 (Generic Sequence 8) , thereby encompassing the morphogenically active sequences of OP-2 and OP-3.
  • each Xaa independently is selected from a group of one or more specified amino acids defined as follows:
  • Xaa at res. 11 (Gin, Leu, Asp, His, Asn or Ser);
  • Generic Sequence 8 (SEQ ID NO: 6) includes all of Generic Sequence 7 and in addition includes the following sequence (SEQ ID NO: 9) at its N-terminus:
  • each "Xaa” in Generic Sequence 8 is a specified amino acid defined as for Generic Sequence 7, with the distinction that each residue number described for Generic Sequence 7 is shifted by five in Generic Sequence 8.
  • Xaa at res.2 (Lys, Arg, Ala or Gin)
  • Xaa at res.3 (Lys, Arg or Met)
  • Xaa at res.4 (His, Arg or Gin)
  • Xaa at res. 5 (Glu, Ser, His, Gly, Arg, Pro, Thr, or Tyr) .
  • useful osteogenic proteins include those defined by Generic Sequences 9 and 10, defined as follows.
  • Generic Sequences 9 and 10 are composite amino acid sequences of the following proteins : human OP-1, human OP-2, human OP-3, human BMP-2, human BMP-3, human BMP-4, human BMP-5, human BMP-6, human BMP-
  • CDMP-3 mouse GDF-7, human BMP-12
  • mouse GDF-3 human GDF-1
  • mouse GDF-1 mouse GDF-1
  • chicken DORSALIN DORSALIN
  • dpp Drosophila SCREW
  • mouse NODAL mouse GDF-8
  • human GDF-8 mouse GDF-
  • Sequence 9 is a 97 amino acid sequence that accommodates the C-terminal six cysteine skeleton and, like Generic Sequence 8, Generic Sequence 10 is a 102 amino acid sequence which accommodates the seven cysteine skeleton.
  • Generic Sequence 10 includes all of Generic Sequence 9 (SEQ ID NO: 7) and in addition includes the following sequence (SEQ ID NO : 9) at its N- terminus :
  • each "Xaa” in Generic Sequence 10 is a specified amino acid defined as for Generic Sequence 9, with the distinction that each residue number described for Generic Sequence 9 is shifted by five in Generic Sequence 10.
  • "Xaa at res. 1 ( Tyr, Phe, His, Arg, Thr, Lys, Gin, Val or Glu)
  • " in Generic Sequence 9 refers to Xaa at res. 6 in Generic Sequence 10.
  • Xaa at res. 2 (Lys, Arg, Gin, Ser, His, Glu, Ala, or Cys) ; Xaa at res.
  • certain currently preferred bone morphogenic polypeptide sequences useful in this invention have greater than 60% identity, preferably greater than 65% identity, with the amino acid sequence defining the preferred reference sequence of hOP-1.
  • useful morphogenic proteins include active proteins comprising pairs of polypeptide chains within the generic amino acid sequence herein referred to as "OPX" (SEQ ID NO: 4), which defines the seven cysteine skeleton and accommodates the homologies between several identified variants of OP-1 and OP-2. As described therein, each Xaa at a given position independently is selected from the residues occurring at the corresponding position in the C-terminal sequence of mouse or human OP- 1 or OP-2. SEQ ID NO: 4
  • Xaa lie Ala Pro Xaa Gly Tyr Xaa Ala Tyr Tyr Cys Glu Gly Glu Cys Xaa Phe Pro 20 25 30 35 Leu Xaa Ser Xaa Met Asn Ala Thr Asn His Ala lie Xaa Gin Xaa Leu Val His Xaa 40 45 50 55 Xaa Xaa Pro Xaa Xaa Val Pro Lys Xaa Cys Cys Ala Pro Thr Xaa Leu Xaa Ala
  • useful osteogenically active proteins have polypeptide chains with amino acid sequences comprising a sequence encoded by a nucleic acid that hybridizes, under low, medium or high stringency hybridization conditions, to DNA or RNA encoding reference morphogen sequences, e . g. , C-terminal sequences defining the conserved seven cysteine domains of OP-1, OP-2, BMP-2, BMP-4, BMP-5, BMP-6, 60A, GDF-3, GDF-6, GDF-7 and the like.
  • reference morphogen sequences e.g. , C-terminal sequences defining the conserved seven cysteine domains of OP-1, OP-2, BMP-2, BMP-4, BMP-5, BMP-6, 60A, GDF-3, GDF-6, GDF-7 and the like.
  • high stringent hybridization conditions are defined as hybridization according to known techniques in 40% formamide, 5 X SSPE, 5 X Denhardt ' s Solution, and 0.1% SDS at 37°C overnight, and washing in 0.1 X SSPE, 0.1% SDS at 50°C.
  • Standard stringent conditions are well characterized in commercially available, standard molecular cloning texts. See, for example, Molecular Cloning A Laboratory Manual , 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D.N. Glover ed., 1985); Oligonucleotide Synthesis (M.J.
  • proteins useful in the present invention generally are dimeric proteins comprising a folded pair of the above polypeptides.
  • Such morphogenic proteins are inactive when reduced, but are active as oxidized homodimers and when oxidized in combination with others of this invention to produce heterodimers.
  • members of a folded pair of morphogenic polypeptides in a morphogenically active protein can be selected independently from any of the specific polypeptides mentioned above.
  • the bone morphogenic proteins useful in the materials and methods of this invention include proteins comprising any of the polypeptide chains described above, whether isolated from naturally-occurring sources, or produced by recombinant DNA or other synthetic techniques, and includes allelic and phylogenetic counterpart variants of these proteins, as well as muteins thereof, and various truncated and fusion constructs. Deletion or addition mutants also are envisioned to be active, including those which may alter the conserved C-terminal six or seven cysteine domain, provided that the alteration does not functionally disrupt the relationship of these cysteines in the folded structure. Accordingly, such active forms are considered the equivalent of the specifically described constructs disclosed herein.
  • the proteins may include forms having varying glycosylation patterns, varying N-termini, a family of related proteins having regions of amino acid sequence homology, and active truncated or mutated forms of native or biosynthetic proteins, produced by expression of recombinant DNA in host cells.
  • the bone morphogenic proteins contemplated herein can be expressed from intact or truncated cDNA or from synthetic DNAs in prokaryotic or eukaryotic host cells, and purified, cleaved, refolded, and dimerized to form morphogenically active compositions.
  • preferred host cells include, without limitation, prokaryotes including E.
  • the invention also relates to an implant device for promoting bone formation, regeneration and repair.
  • the implant device comprises the bone precursor composition material of the invention, and optionally at least one additional agent selected from a bioactive agent or a binder .
  • the implant device comprising a cement matrix formed from the bone precursor composition serves as a temporary scaffold and substratum for recruitment of migratory progenitor cells, and as a base for their subsequent anchoring and proliferation.
  • the implant device comprises the bone precursor composition and a bioactive agent, which is dispersed or absorbed in the bone precursor composition.
  • the cement matrix formed from the bone precursor composition provides a delivery or support system for the bioactive agent, which is released over time at the implantation site as the bone precursor composition is slowly absorbed.
  • the bioactive agent is encapsulated in the pore-forming agent. The resorption pf the pore-forming agent and the gradual release of the bioactive agent provides a sustained release system.
  • the dosage and rate of delivery of the bioactive agent may be controlled based on the nature of the cement, the nature of the pore-forming agent and the nature of the binding interaction between the bioactive agent, the cement and pore-forming agent.
  • the bioactive agent is a bone morphogenic protein or a nucleic acid molecule that encodes BMP.
  • the BMP is OP-1.
  • the cement matrix formed by the bone precursor composition can protect the BMP from non-specific proteolysis, and can accommodate each step of the cellular responses involved in progenitor cell induction during tissue development.
  • the osteogenic protein diffuses out of the cement matrix into the implantation site and permits influx and efflux of cells.
  • the osteogenic protein induces the progenitor cells to differentiate and proliferate.
  • Progenitor cells may migrate into the cement matrix and differentiated cells may move out of the cement matrix into the implant site.
  • the sequential cellular reactions in the interface of the cement matrix/osteogenic protein implants include: binding of fibrin and fibronectin to implanted cement matrix, migration and proliferation of mesenchymal cells, differentiation of the progenitor cells into chondroblasts, cartilage formation, cartilage calcification, vascular invasion, bone formation, remodeling, and bone marrow differentiation.
  • the preferred osteogenic device with the bone precursor composition can be used in various orthopedic, periodontal, and reconstructive procedures.
  • the implant device may also comprise a binder in an admixture with the bioactive agent and/or bone precursor composition material .
  • the binder is added to regulate the moldability of the composition to fit a defect site or to take the form of a new tissue.
  • the implant material may be molded into a variety of shapes depending on the tissue defect site to which it is administered.
  • the implant material may be injected. For example, healing of closed fresh fractures can be accelerated through single minimally invasive percutaneous injections of implanted material. See, e . g. , Blokhuis et al . Biomaterials 22, pp.
  • the implant material may have a preferred shape which allows implantation at a defect site.
  • the moldable cement matrix formed from the bone precursor composition can be held in place by the surrounding tissue or masticated muscle. It is preferred to shape the composition to span a tissue defect and to take the desired form of the new tissue. In the case of bone repair of a non-union defect, for example, it is desirable to use dimensions that span the non-union. Rat studies show that the newly synthesized bone has the dimensions of the implanted device.
  • the material may be used for subcutaneous or intramuscular implants. In bone formation procedures, the material is slowly absorbed by the body and is replaced by bone in the shape of or very nearly the shape of the implant .
  • the bone precursor composition material of the present invention may be used in a prosthetic device.
  • the prosthetic device comprises a surface region that can be implanted adjacent to a target tissue of a mammal, and a composition that is disposed on the surface region.
  • the prosthetic devices will be useful for repairing orthopedic defects, injuries or anomalies in the treated mammal.
  • the mammal is a human patient.
  • the prosthetic device may be made from a material comprising metal, ceramic or polymer composite material .
  • Preferred devices comprise a load- bearing core selected from Co-Cr-Mo alloys, titanium alloys and stainless steel.
  • Preferred prosthetic devices are selected from the group consisting of a hip device, a fusion cage and a maxillofacial device.
  • the composition comprises the bone precursor composition of the invention, and optionally, one or more agents selected from the group consisting of a bioactive agent or a binder dispersed in the bone precursor composition.
  • the bioactive agent is encapsulated in the pore-forming agent.
  • the bioactive agent is a BMP or nucleic acid comprising a sequence encoding BMP, more preferably, OP-1.
  • the composition may act as a coating for synthetically constructed bone material, such as for an artificial hip, replacement of diseased bone, correction of defects, or anchoring teeth.
  • Osteogenic protein-coated prosthetic devices may enhance the bond strength between the prosthesis and existing bone (Rueger et al., U. S. Patent No. 5,344,654, incorporated herein by reference).
  • the prosthetic device is coated with a hydroxyapatite or beta-tricalcium phosphate material to facilitate the integration of the composition of this invention onto the prosthetic device. This embodiment is particularly advantageous when there is a lack of bone mass around the prosthetic device.
  • the composition is disposed on the surface of the implant in an amount sufficient to promote enhanced tissue growth into the surface. The amount of the composition sufficient to promote enhanced tissue growth may be determined empirically by those of skilled in the art using bioassays described in Rueger et al., U. S. patent No.
  • the composition can be used in ligament repair such as anterior cruciate ligament fixation or ligament attachment in the appendicular system to assist in the integration of ligament and bone.
  • the composition is applied to the clinical procedure of total joint arthroplasty in hips, knees, elbows and other joints, wherein a diseased or damaged natural joint is replaced by a prosthetic joint.
  • a prosthetic joint For example, in a total hip arthroplasty, an acetabular cup is inserted with the composition in the acetabular socket of the pelvis to replace the natural acetabulum. The cup is held in place by the composition and secured by fixation screws. Generally, the cavity or socket conforms to the outer surface of the acetabular cup.
  • the composition can also be applied to total joint revision surgery, to strengthen the bondage between joint prosthetic devices and the bone .
  • the composition is applied to a clinical procedure called vertebroplasty.
  • the composition is injected into the interior of a vertebral body. This method is used in the treatment of osteoporosis to increase the density of bone .
  • the prosthetic device is selected from the group consisting of a fusion cage, a dowel and other devices having a pocket or chamber, such as an interbody fusion for containing the composition of the present invention.
  • the interbody fusion device is produced from material selected from the group consisting of titanium, PEEK (pol (etheretherketone) ) and allograft.
  • the interbody fusion in the cervical, thoracic and lumbar spine can be administered via an anterior or posterior approach.
  • the composition of this invention can be used without an associated interbody device to achieve interbody fusion.
  • the fusion cages are placed into the intervertebral space left after the removal of a damaged spinal disc to eliminate local motion and to participate in vertebral to vertebra bony fusion.
  • the fusion cages are in the form of a cylindrical hollow member having an outside diameter larger than the space between two adjacent vertebrae to be fused.
  • the interior space within the cylindrical hollow implant can be filled with the composition of this invention.
  • the cylindrical implants can also include a threaded exterior to permit threaded insertion into a tapped bore formed in the adjacent vertebrae. Alternatively, some fusion implants have been designed to be impacted into the intradiscal space. As described in U.S. patent No.
  • the fusion device includes opposite end pieces with an integral central element.
  • the central element has a much smaller diameter so that the fusion device forms an annular pocket around the central element.
  • the composition of this invention can be disposed within the annular pocket between the opposite end pieces.
  • the prosthetic device is used for repair of osseous and discoligamentous instability.
  • the composition of this invention may be applied to the intervertebral area, resulting in superior fusion and consequently achieving definitive stabilization of a traumatized motor segment via a single dorsal approach.
  • This application may eliminate the need to undergo a second operation for fractures of the thoracolumbar spine, which, at present, is often necessary but involves additional high risks.
  • this method avoids the problems associated with transplantation of autogenous cancellous bone and its associated risk of high morbidity. See, e . g. , Rueger et al . , Orthopade , 27, pp. 72-79 (1998).
  • the prosthetic device is a maxillofacial device. Maxillofacial devices are applied externally to correct facial defects resulting from cancer surgery, accidents, congenital deformities. In order to restore the masticatory deficiencies, a patient with marginal bone mass is first treated with the composition of this invention to pack and build up the surgical site.
  • a maxillofacial anchoring and distracting system as illustrated in U.S. patent No. 5,899,940, can be applied to increase the existing bone quality.
  • Fixation devices such as a standard threaded bone screw and simple pin point tack or self-locking and threaded bone tack screw device (U.S. patent No.
  • the invention also provides a method for promoting in vivo integration of an implantable prosthetic device of this invention into a target tissue of a mammal comprising the steps of a) providing on a surface of the prosthetic device a composition comprising the bone precursor composition, optionally, at least one bioactive agent or a binder, and b) implanting the device in a mammal at a locus where the target tissue and the surface of the prosthetic device are maintained at least partially in contact for a time sufficient to permit tissue growth between the target tissue and the device.
  • the invention also provides a method of inducing bone formation or repair in a mammal .
  • the mammal is preferably a human patient .
  • the method comprises the step of implanting in the defect site of a mammal a composition comprising the bone precursor composition of the invention.
  • the composition may further comprise a binder and/or a bioactive agent.
  • the bioactive agent is encapsulated in the pore-forming agent.
  • the defect can be an endochondral defect, an osteochondral defect or a segmental defect.
  • the method can also be applied to other defects which include, but are not limited to, nonunion fractures; bone cavities; tumor resection; fresh fractures (distracted or undistracted) ; cranial, maxillofacial and facial abnormalities, for example, in facial skeletal reconstruction, specifically, orbital floor reconstruction, augmentation of the alveolar ridge or sinus, periodontal defects and tooth extraction socket; cranioplasty, genioplasty, chin augmentation, palate reconstruction, and other large bony reconstructions; vertebroplasty, interbody fusions in the cervical, thoracic and lumbar spine and posteriolateral fusions in the thoracic and lumbar spine; in osteomyelitis for bone regeneration; appendicular fusion, ankle fusion, total hip, knee and joint fusions or arthroplasty; correcting tendon and/or ligamentous tissue defects such as, for example, the anterior, posterior, lateral and medial ligaments of the knee, the patella and achilles tendons, and the like as well as those defects resulting from diseases
  • the invention also provides a method of delivering a bioactive agent at a site requiring bone formation comprising the step of implanting the bone precursor composition and a bioactive agent at the defect site of a mammal.
  • the method of delivering the bone precursor composition may further include a binder.
  • the bioactive agent is encapsulated in the pore-forming agent.
  • the bioactive agent belongs to the bone morphogenic protein family.
  • the bioactive agent is a nucleic acid molecule comprising a sequence encoding a BMP.
  • the nucleic acid is trapped in a carrier.
  • the bioactive agent is a bone cell or a bone cell expressing BMP.
  • the delivery of the bioactive agent is sustained release.
  • the pore-forming agent is preferably a biocompatible and non-immunogenic polymer, more preferably, PLGA.
  • the bioactive agent is preferably OP-1.
  • the release rate of the bioactive agent can be controlled by altering the molecular weight of the PLGA.
  • the degradation of PLGA commences when water penetrates the cement matrix to hydrolyze long polymer chains into short water soluble fragments. There is a reduction in molecular weight of the PLGA without loss in its physical properties. Gradually, further erosion of the polymer leads to the disruption of the polymer, thereby releasing the bioactive agent.
  • the rate of release for OP-1 is one to six weeks. Examples
  • the PLGA polymer microspheres were supplied from Alkermes, Inc.
  • the calcium sulfate pore-forming agent was prepared by hydrating calcium sulfate hemihydrate. The wet calcium sulfate mass was passed through a sieve to form granules, which were hardened after drying, and resieved to break any agglomerates.
  • Microsphere PLGA beads having a particle size of 75-150 ⁇ m were mixed with BoneSource hydroxyapatite cement powder (containing tetracalcium phosphate and dicalcium phosphate anhydrous) at different ratios
  • 0.2 N HCl acid for 24 hours to conduct a rapid simulation of the in vivo resorption activity.
  • 5 ml of 0.2 N HCl was added to each implant that was placed in a 5 ml glass vial. The acid surface covered the implant completely. The vial was stoppered and placed on an automatic shaker with moderate shaking. The appearance of the implants was observed periodically.
  • This assay consists of implanting the composition of this invention in subcutaneous sites in recipient rats under ether anesthesia. Male Long-Evans rats, aged 28-32 days, may be used. A vertical incision (1 cm) is made under sterile conditions in the skin over the thoracic region, and a pocket is prepared by blunt dissection. Approximately 25 mg of the test sample is implanted deep into the pocket and the incision is closed with a metallic skin clip. The day of implantation is designated, as day one of the experiment . Implants are removed at varying times thereafter (i.e. 12 days, 18 days) . The heterotrophic site allows for the study of bone induction without the possible ambiguities resulting from the use of orthotropic sites.
  • Bone growth is determined biochemically by calcium content of the implant. Calcium content, is proportional to the amount of bone formed in the implant. Bone formation therefore is calculated by determining the calcium content of the implant in rats and is expressed as "bone forming units," where one bone forming unit represents the amount of protein that is needed for half maximal bone forming activity of the implant . Bone induction exhibited by intact demineralized rat bone matrix is considered to be the maximal bone differentiation activity for comparison purposes in this assay.
  • Histological sectioning and staining is a preferred method to determine the extent of osteogenesis in the implants. Implants are fixed in Bouins Solution, embedded in paraffin, and cut into 6-8 ⁇ m sections. Staining with toluidine blue or hemotoxylin/eosin demonstrates clearly the ultimate development of endochondral bone. Twelve-day implants are usually sufficient to determine whether the implants contain newly-induced bone.
  • Alkaline phosphatase (AP) activity may be used as a marker for osteogenesis.
  • the enzyme activity may be determined spectrophotometrically after homogenization of the implant. The activity in vivo peaks subsequently and thereafter slowly declines. Implants showing no bone development by histology have little or no alkaline phosphatase activity under these assay conditions.
  • the assay is useful for quantification and obtaining an estimate of bone formation quickly after the implants are removed from the rat . Alternatively, the amount of bone formation can be determined by measuring the calcium content of the implant .
  • Gene expression patterns that correlate with endochondral bone or other types of tissue formation can also be monitored by quantitating mRNA levels using procedures known to those of skill in the art such as Northern Blot analysis.
  • Such developmental gene expression markers may be used to determine progression through tissue differentiation pathways after treatment with osteogenic protein. These markers include osteoblastic-related matrix proteins such as procollagen 2 (I), procollagen 1 (I), procollagen 1 (III), osteonectin, osteopontin, biglycan, and alkaline phosphatase for bone regeneration (see e . g. , Suva et al . , J. Bone Miner. Res., 8, pp. 379-88 (1993) ; Benayahu et al . , J. Cell. Biochem. , 56, pp. 62-73 (1994)).
  • the periosteum was incised and maintained intact for surgical closure if possible.
  • Three transverse holes were created in the metaphysis. The first and most superior was created approximately 2 cm below the articular surface of the tibia. The defects were created so as to form a line oriented with the long axis of the bone. Implants were spaced at 1.6 cm intervals measured center-to-center.
  • Materials were harvested at four and eight weeks post-treatment. Animals were euthanised with pentobarbital 75-100 mg/kg. The proximal tibia was taken and cut to best allow for tissue fixation. Specimens were fixed in 10% neutral buffered Formalin. Specimens were cut, if feasible, so as to capture all implant sites in a single specimen. Following fixation, specimens were decalcified, embedded in plastic and sectioned in longitudinal orientation using Exackt technique and ground to appropriate section thickness for histologic interpretation.
  • Radiographic assessment and histologic evaluation of all implant sites were made at four and eight weeks post-operative .
  • Anterior posterior radiographs were taken so as to best image all three defects simultaneously and view the cylindrical defects from the side.
  • Qualitative histologic descriptions identified new bone formation, residual implant material and any evidence of pathologic response . Images were captured for each specimen and scores presented for bone formation, acute and chronic inflammation and residual matrix.
  • Proximal tibia sections contained three defects. These sections were gross macro-cut so that all three defects were contained in a single section. Based on gross section observations, clinical assays, and faxitron x-rays of this section, the section was considered representative of the sample. This orientation allowed the evaluation of the periosteal reaction overlying the defects and intramedullary response to the test materials. Specimens were evaluated from 4 and 8 week explants ( Figures 1-6) .
  • Tissues from the sheep model bioassay were evaluated for bone formation using paraffin sections and hematoxylin and eosin stain. Tibial specimens were sectioned so as to isolate implant sites in the proximal, middle and distal sites from animals. These explants were decalcified, embedded in paraffin, sectioned and stained with hematoxylin and eosin. [0143] Sections were viewed using light microscopy and interpreted for bone formation. For specimens stratified in bone formation, the response from the cortical level was robust and deep, and the response was modest in the medullary compartment. Due to this stratification, the level extending from the endosteal cortex to a level 2-3 mm deep was evaluated.
  • a femoral osteotomy defect is surgically prepared. Without further intervention, the simulated fracture defect would consistently progress to non-union.
  • the effects of the composition and devices of this invention implanted into the created bone defects are evaluated by the following study protocol. [0145] Briefly, the procedure is as follows: Sixteen adult cats each weighing less than 10 lbs. undergo unilateral preparation of a 1 cm bone defect in the right femur through a lateral surgical approach. In other experiments, a 2 cm bone defect may be created. The femur is immediately internally fixed by lateral placement of an 8-hole plate to preserve the exact dimensions of the defect.
  • group I is a negative control group with no test material
  • group II is implanted with the cement matrix formed from the bone precursor composition
  • group III is implanted with the cement matrix formed from the bone precursor composition and an osteogenic protein.
  • All animals are allowed to ambulate ad libi tum within their cages post-operatively. All cats are injected with tetracycline (25 mg/kg subcutaneously (SQ) each week for four weeks) for bone labeling.
  • SQ subcutaneously
  • Excised test and normal femurs may be immediately studied by bone densitometry, or wrapped in two layers of saline-soaked towels, placed into sealed plastic bags, and stored at -20° C until further study. Bone repair strength, load-to-failure, and work-to- failure are tested by loading to failure on a specially designed steel 4-point bending jig attached to an Instron testing machine to quantitate bone strength, stiffness, energy absorbed and deformation to failure. The study of test femurs and normal femurs yields the bone strength (load) in pounds and work-to-failure in joules. Normal femurs exhibit a strength of 96 (+/- 12) pounds. Osteogenic device-implanted femur strength should be corrected for surface area at the site of fracture (due to the "hourglass" shape of the bone defect repair) . With this correction, the result should correlate closely with normal bone strength.
  • Ulnae defects are followed for the full course of the eight week study in each group of rabbits.
  • the marrow cavity of the 1.5 cm ulnar defect is packed with osteogenic protein in a cement matrix formed from the bone precursor composition.
  • the bones are allografted in an intercalary fashion. Negative control ulnae are not healed by eight weeks and reveal the classic "ivory" appearance.
  • the osteogenic protein-treated implants "disappear" radiographically by four weeks with the start of remineralization by six to eight weeks. These allografts heal at each end with mild proliferative bone formation by eight weeks. This type of device serves to accelerate allograft repair.
  • the rabbit model may also be used to test the efficacy of and to optimize conditions under which a particular composition of this invention can induce local bone formation.
  • This assay is performed essentially as described in Cook et al . , Clinical Orthopaedics and Related Research, 301, pp. 302-112 (1994), which is incorporated herein by reference) . Briefly, an ulnar segmental defect model is used to evaluate bone healing in 35-45 kg adult male dogs. Experimental composites comprising 500 mg of bone precursor composition are reconstituted with varying amounts of OP-1. Any osteogenic protein may be used in place of OP-1 in this assay. Implantations at defect sites are performed with one carrier control and with the experimental series of OP-1 being tested. Mechanical testing is performed on ulnae of animals receiving composites at 12 weeks after implantation.
  • Radiographs of the forelimbs are obtained weekly until the animals are sacrificed at either 12 or 16 postoperative weeks. Histological sections are analyzed from the defect site and from adjacent normal bone .
  • EXAMPLE 9 Monkey Ulnar and Tibial Defect Bioassay For Bone Repair
  • This bone healing assay in African green monkeys is performed essentially as described in Cook et al . , J. Bone and Joint Surgery, 77A, pp. 734-50 (1995), which is incorporated herein by reference. Briefly, a 2.0 cm osteoperiosteal defect is created in the middle of the ulnar shaft and filled with an implant comprising bone precursor composition matrices containing OP-1. Experimental composites comprising bone precursor composition matrices reconstituted with varying amounts of OP-1 are used to fill 2.0 cm osteoperiosteal defects created in the diaphysis of the tibia. Any osteogenic protein may be used in place of OP-1 in this assay.
  • Implantations at defect sites are performed with one carrier control and with the experimental series of OP-1 being tested. Mechanical testing is performed on ulnae and tibia of animals receiving composites. Radiographs and histological sections are analyzed from the defect sites and from adjacent normal bone as described in Cook et al .
  • This fracture healing assay in sheep is performed essentially as described in Blokhius et al . , Biomaterials, 22, pp. 725-730 (2001), which is incorporated herein by reference.
  • a closed midshaft fracture is created in the left tibia of adult female goats with a custom-made three point bending device. The fractures are stabilized with an external fixator, which is placed at the lateral side of the tibia.
  • Three different types of materials are implanted in the goat defects via injection: group I is a negative control group with no test material; group II is implanted with the cement matrix formed from the bone precursor composition; and group III is implanted with the cement matrix formed from the bone precursor composition and an osteogenic protein.
  • the injections are given under aseptic conditions and fluroscopy is used to ascertain that the injected material was placed in the fracture gap.
  • Mechanical testing (4-point non-destructive bending test) is performed on the animals receiving composites at two weeks and four weeks. After the mechanical testing, anterior, posterior, lateral, and medial slices of the fracture gap are sawn to perform radiographs and histological sections.
  • a motor segment of the spine is a functional unit consisting of two vertebral bodies lying one above the other, and an intervertebral disc.
  • a trial group consists of 12 sheep. Two control groups of 12 sheep each are used. The surgical area at the inferior lumbar spine is prepared after introduction of general anesthesia and placing the animals in prone position. A skin incision of about 12 cm in length above the spinous processes of the inferior lumbar spine is made. After transsection of the subcutis and fascia, the back muscles are moved to the side. [0159] Intubation anesthesia is applied by
  • Further dosage can be administered as needed.
  • the sedation requires placement of an intravenous indwelling catheter after puncturing an ear vein.
  • the anesthesia is introduced through the catheter by providing 3-5 mg of
  • the animals are ventilated using oxygen (30%) , nitrous oxide (laughing gas) and isoflurane (Isofluran ) .
  • oxygen (30%)
  • nitrous oxide nitrous oxide
  • Isofluran isoflurane
  • the analgesic fentanyl dihydrogen citrate (Fentanyl ) having a dosage 0.2-0.4 mg, is administered.
  • relaxation is achieved by administration of atracurium besilate (Atracurium ® ) at a dosage of 0.5mg/kg of body weight .
  • test samples include cement matrix and osteogenic protein in varying concentrations.
  • first control group that consists of 12 sheep, only the cement matrix is applied.
  • second control group autologous spongiosa is administered instead of the composition of this invention.
  • the internal fixator is installed completely. The type of the internal fixator as well as the necessary instrumentation is the same as those used in humans. Accordingly, the surgical procedure is standardized and well known to the skilled practitioner. Drains are placed and the wound is closed using absorbable suture for fascia and subcutis as well as skin staples.
  • an x-ray image amplifier is available for intraoperative fluoroscopy. This facilitates exact orientation during the execution of the above steps.
  • Clinical Observations [0165] Daily neurologic examinations are performed to evaluate the gait of the animals as well as neurological deficits that may occur postoperatively. Operative wounds are closely examined each day. Body weights are measured preoperatively and at the time of euthanasia.
  • A indicates a big, solid trabeculated bilateral fusion mass (definitely solid) ;
  • B a big, solid unilateral fusion mass with a small contralateral fusion mass (possibly solid) ;
  • C a small, thin bilateral fusion mass with an apparent crack (probably not solid) ;
  • D bilateral resorption of the graft or fusion mass with an obvious bilateral pseudarthrosis (definitely not solid) .
  • computerized tomography scans are performed to assess the fusion mass in cross sections and in saggital-plane reconstructions. For each fusion mass, approximately forty sequential computerized tomography scans are made with use of two-millimeter slice intervals and subsequent reconstruction in the saggital plane under consistent magnification and radiographic conditions.
  • Biomechanical Testing [0168] Four specimens of each group are evaluated biomechanically. After radiographic analysis, all muscles are carefully removed while maintaining the ligamentous and bony structures. The spines are frozen at -20°C. For each of these specimens, the upper half of the upper vertebra and the lower half of the lower vertebra of the motion segment L4/L5 are embedded in polymethylmethacrylate (Technovit 3040; Heraeus Kulzer GmbH, Wehrheim/Ts, Germany) . Each specimen is then fixed and tested without preload in a spine tester in a non-destructive testing mode.
  • Polymethylmethacrylate Technovit 3040; Heraeus Kulzer GmbH, Wehrheim/Ts, Germany
  • Alternating sequences of flexion/extension, axial right/left rotation, and right/left lateral bending moments are applied continuously at a constant rate of 1.7 degrees/second by stepper motors integrated in the gimbal of the spine tester. Two precycles are applied to minimize the effect of the viscous component in the viscoelastic response, and data will be collected on the third cyle. Range of motion, neutral zone, and two stiffness parameters are determined from the resulting load-deformation curves.
  • Fluorochrome sequential analysis is then performed by Fluorescence microscopy on the specimens under UV light for qualitative and quantitative dynamic evaluation.

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Abstract

Composition précurseur d'os contenant un mélange de ciment et un agent porogène est conçue pour exécuter un implant osseux. Cet agent porogène, de préférence, présente une dimension de particule de 20-500 microns. Dans un mode de réalisation préféré, la proportion de cet agent porogène est 7-40 % (w/w). Cette composition peut également contenir un agent bioactif, de préférence, une protéine morphogénique osseuse ou un acide nucléique codant ladite protéine (BMP) et encapsulé dans l'agent porogène. On peut moduler les caractéristiques de moulage de cette composition par l'intermédiaire d'un apport de liant. L'invention concerne également une trousse et un implant comprenant ladite composition précurseur d'os. Elle concerne également un dispositif prothétique implantable composé d'un implant prothétique possédant une zone superficielle dans laquelle est placé le matériau précurseur d'os. Cette trousse et ces dispositifs peuvent, de plus, contenir un ou plusieurs éléments supplémentaires, tels qu'un agent bioactif et un liant. Elle concerne enfin des procédés servant à induire la formation osseuse et à administrer cet agent bioactif.
PCT/US2002/029966 2001-09-21 2002-09-20 Agents porogenes pour ciments orthopediques WO2003024316A2 (fr)

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AU2002327007A AU2002327007B2 (en) 2001-09-21 2002-09-20 Pore-forming agents for orthopedic cements
CA002460843A CA2460843A1 (fr) 2001-09-21 2002-09-20 Agents porogenes pour ciments orthopediques
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Cited By (33)

* Cited by examiner, † Cited by third party
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US7211266B2 (en) 2002-03-29 2007-05-01 Wright Medical Technology, Inc. Bone graft substitute composition
US7291179B2 (en) 2002-06-24 2007-11-06 Wright Medical Technology, Inc. Bone graft substitute composition
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US7524335B2 (en) 1997-05-30 2009-04-28 Smith & Nephew, Inc. Fiber-reinforced, porous, biodegradable implant device
US20090148487A1 (en) * 2005-12-14 2009-06-11 Scil Technology Gmbh Moldable biomaterial for bone regeneration
US20100068243A1 (en) * 2006-08-24 2010-03-18 Graftys Macroporous and highly resorbable apatitic calcium-phosphate cement
US20100166867A1 (en) * 2008-11-19 2010-07-01 Adocia Novel administration form of osteogenic protein complexes
US7749268B2 (en) 2004-05-26 2010-07-06 Warsaw Orthopedic, Inc. Methods for treating the spine
US7754246B2 (en) 2005-09-09 2010-07-13 Wright Medical Technology, Inc. Composite bone graft substitute cement and articles produced therefrom
US7766972B2 (en) 2004-10-22 2010-08-03 Wright Medical Technology, Inc. Synthetic, malleable bone graft substitute material
WO2010138637A2 (fr) 2009-05-26 2010-12-02 The Regents Of The University Of California Peptide de la fibromoduline
US7857860B2 (en) 2003-04-30 2010-12-28 Therics, Llc Bone void filler and method of manufacture
US8025903B2 (en) 2005-09-09 2011-09-27 Wright Medical Technology, Inc. Composite bone graft substitute cement and articles produced therefrom
US8101198B2 (en) 2006-07-26 2012-01-24 The Regents Of The University Of California Osteogenic enhancer composition
WO2012024573A2 (fr) 2010-08-19 2012-02-23 The Regents Of The University Of California Compositions comprenant des cellules souches périvasculaires et la protéine nell-1
US8124118B2 (en) 2003-10-22 2012-02-28 Lidds Ab Composition comprising biodegradable hydrating ceramics for controlled drug delivery
EP2450064A1 (fr) * 2010-10-19 2012-05-09 National Cheng Kung University Formulation de ciment osseux et composites durcis de ciment osseux préparés avec cette formulation
US8545866B2 (en) 2004-10-29 2013-10-01 Smith & Nephew, Inc. Bioabsorbable polymers
CN103394120A (zh) * 2013-07-31 2013-11-20 华南理工大学 一种磷酸钙基复合微球支架及其制备方法
WO2014091469A1 (fr) * 2012-12-14 2014-06-19 Ossdsign Ab Compositions formant un ciment, ciments de monétite, implants et procédés permettant de corriger les défauts osseux
US9206080B2 (en) 2008-11-12 2015-12-08 Ossdsign Ab Hydraulic cements, methods and products
US9486558B2 (en) 2003-03-27 2016-11-08 Locate Therapeutics Limited Porous matrix
WO2017051356A1 (fr) * 2015-09-23 2017-03-30 Ossdsign Ab Composition formant un ciment, ciments d'apatite, implants et procédés de correction d'anomalies osseuses
CN106806403A (zh) * 2017-02-17 2017-06-09 福建康是美生物科技有限公司 一种增加骨密度的中药复方组合物及其制备方法
EP2588154B1 (fr) 2010-07-02 2017-10-18 Agnovos Healthcare, LLC Composition contenant des poudres de phosphate ou de sulfate de calcium ou de dbm utilisée pour traiter des pathologies osseuses dégénératives
CN108114323A (zh) * 2018-01-24 2018-06-05 广西医科大学 一种多孔可注射的磷酸钙骨水泥复合物
US10207027B2 (en) 2012-06-11 2019-02-19 Globus Medical, Inc. Bioactive bone graft substitutes
WO2019048697A1 (fr) 2017-09-11 2019-03-14 Bone Support Ab Biomatériau de cryogel composite macroporeux et microporeux destiné à être utilisé dans la régénération osseuse
EP3520804A1 (fr) 2006-11-07 2019-08-07 The Regents of The University of California Composition pour cartilage
CN111790004A (zh) * 2020-06-17 2020-10-20 天津市康婷生物工程集团有限公司 一种通用型载药钙磷骨水泥多孔支架制备方法
WO2021034406A1 (fr) * 2019-08-19 2021-02-25 Lin, Jiin-Huey, Chern Dispositif hémostatique et procédé associé
US10973949B2 (en) 2013-12-13 2021-04-13 Agnovos Healthcare, Llc Multiphasic bone graft substitute material
US11395864B2 (en) 2016-06-10 2022-07-26 Dsm Ip Assets B.V. Settable bone void filler

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CN107303397B (zh) * 2016-04-20 2019-10-01 中国科学院化学研究所 一种具有生物活性的可注射复合骨水泥及其制备方法和用途
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US7524335B2 (en) 1997-05-30 2009-04-28 Smith & Nephew, Inc. Fiber-reinforced, porous, biodegradable implant device
US8968465B2 (en) 2002-03-29 2015-03-03 Wright Medical Technology, Inc. Bone graft substitute composition
US7211266B2 (en) 2002-03-29 2007-05-01 Wright Medical Technology, Inc. Bone graft substitute composition
US8657952B2 (en) 2002-03-29 2014-02-25 Wright Medical Technology, Inc. Bone graft substitute composition
US7291179B2 (en) 2002-06-24 2007-11-06 Wright Medical Technology, Inc. Bone graft substitute composition
US7658768B2 (en) 2002-06-24 2010-02-09 Wright Medical Technology, Inc. Bone graft substitute composition
US10232087B2 (en) 2003-03-27 2019-03-19 Locate Therapeutics Limited Porous matrix
US9486558B2 (en) 2003-03-27 2016-11-08 Locate Therapeutics Limited Porous matrix
US7857860B2 (en) 2003-04-30 2010-12-28 Therics, Llc Bone void filler and method of manufacture
US9034359B2 (en) 2003-10-22 2015-05-19 Lidds Ab Composition comprising biodegradable hydrating ceramics for controlled drug delivery
US8124118B2 (en) 2003-10-22 2012-02-28 Lidds Ab Composition comprising biodegradable hydrating ceramics for controlled drug delivery
JP2007536044A (ja) * 2004-05-03 2007-12-13 サントル ナスィオナル ド ラ ルシェルシュ スィアンティフィク 骨置換物として有用な注入可能セメント用組成物
US8343221B2 (en) 2004-05-26 2013-01-01 Warsaw Orthopedic, Inc. Methods for treating the spine
US7749268B2 (en) 2004-05-26 2010-07-06 Warsaw Orthopedic, Inc. Methods for treating the spine
US7766972B2 (en) 2004-10-22 2010-08-03 Wright Medical Technology, Inc. Synthetic, malleable bone graft substitute material
US9387274B2 (en) 2004-10-29 2016-07-12 Smith & Nephew, Inc. Bioabsorbable polymers
US8545866B2 (en) 2004-10-29 2013-10-01 Smith & Nephew, Inc. Bioabsorbable polymers
US9173981B2 (en) 2004-10-29 2015-11-03 Smith & Nephew, Inc. Bioabsorbable polymers
US8025903B2 (en) 2005-09-09 2011-09-27 Wright Medical Technology, Inc. Composite bone graft substitute cement and articles produced therefrom
US9180224B2 (en) 2005-09-09 2015-11-10 Agnovos Healthcare, Llc Composite bone graft substitute cement and articles produced therefrom
US8685465B2 (en) 2005-09-09 2014-04-01 Agnovos Healthcare, Llc Composite bone graft substitute cement and articles produced therefrom
US8685464B2 (en) 2005-09-09 2014-04-01 Agnovos Healthcare, Llc Composite bone graft substitute cement and articles produced therefrom
US7754246B2 (en) 2005-09-09 2010-07-13 Wright Medical Technology, Inc. Composite bone graft substitute cement and articles produced therefrom
US20090148487A1 (en) * 2005-12-14 2009-06-11 Scil Technology Gmbh Moldable biomaterial for bone regeneration
US8101198B2 (en) 2006-07-26 2012-01-24 The Regents Of The University Of California Osteogenic enhancer composition
US20100068243A1 (en) * 2006-08-24 2010-03-18 Graftys Macroporous and highly resorbable apatitic calcium-phosphate cement
US9642939B2 (en) * 2006-08-24 2017-05-09 Graftys Macroporous and highly resorbable apatitic calcium-phosphate cement
EP3520804A1 (fr) 2006-11-07 2019-08-07 The Regents of The University of California Composition pour cartilage
US9206080B2 (en) 2008-11-12 2015-12-08 Ossdsign Ab Hydraulic cements, methods and products
US9540280B2 (en) 2008-11-12 2017-01-10 Ossdsign Ab Hydraulic cements, methods and products
US20100166867A1 (en) * 2008-11-19 2010-07-01 Adocia Novel administration form of osteogenic protein complexes
US8546356B2 (en) * 2008-11-19 2013-10-01 Adocia Administration form of osteogenic protein complexes
WO2010138637A2 (fr) 2009-05-26 2010-12-02 The Regents Of The University Of California Peptide de la fibromoduline
EP2588154B1 (fr) 2010-07-02 2017-10-18 Agnovos Healthcare, LLC Composition contenant des poudres de phosphate ou de sulfate de calcium ou de dbm utilisée pour traiter des pathologies osseuses dégénératives
WO2012024573A2 (fr) 2010-08-19 2012-02-23 The Regents Of The University Of California Compositions comprenant des cellules souches périvasculaires et la protéine nell-1
EP2450064A1 (fr) * 2010-10-19 2012-05-09 National Cheng Kung University Formulation de ciment osseux et composites durcis de ciment osseux préparés avec cette formulation
US10792397B2 (en) 2012-06-11 2020-10-06 Globus Medical, Inc. Bioactive bone graft substitutes
US10207027B2 (en) 2012-06-11 2019-02-19 Globus Medical, Inc. Bioactive bone graft substitutes
WO2014091469A1 (fr) * 2012-12-14 2014-06-19 Ossdsign Ab Compositions formant un ciment, ciments de monétite, implants et procédés permettant de corriger les défauts osseux
AU2013358613B9 (en) * 2012-12-14 2017-11-02 Ossdsign Ab Cement-forming compositions, monetite cements, implants and methods for correcting bone defects
US9913931B2 (en) 2012-12-14 2018-03-13 Ossdsign Ab Cement-forming compositions, monetite cements, implants and methods for correcting bone defects
AU2013358613B2 (en) * 2012-12-14 2017-08-24 Ossdsign Ab Cement-forming compositions, monetite cements, implants and methods for correcting bone defects
CN103394120B (zh) * 2013-07-31 2015-01-28 华南理工大学 一种磷酸钙基复合微球支架及其制备方法
CN103394120A (zh) * 2013-07-31 2013-11-20 华南理工大学 一种磷酸钙基复合微球支架及其制备方法
US10973949B2 (en) 2013-12-13 2021-04-13 Agnovos Healthcare, Llc Multiphasic bone graft substitute material
WO2017051356A1 (fr) * 2015-09-23 2017-03-30 Ossdsign Ab Composition formant un ciment, ciments d'apatite, implants et procédés de correction d'anomalies osseuses
US11395864B2 (en) 2016-06-10 2022-07-26 Dsm Ip Assets B.V. Settable bone void filler
CN106806403A (zh) * 2017-02-17 2017-06-09 福建康是美生物科技有限公司 一种增加骨密度的中药复方组合物及其制备方法
WO2019048697A1 (fr) 2017-09-11 2019-03-14 Bone Support Ab Biomatériau de cryogel composite macroporeux et microporeux destiné à être utilisé dans la régénération osseuse
CN108114323A (zh) * 2018-01-24 2018-06-05 广西医科大学 一种多孔可注射的磷酸钙骨水泥复合物
WO2021034406A1 (fr) * 2019-08-19 2021-02-25 Lin, Jiin-Huey, Chern Dispositif hémostatique et procédé associé
CN114555098A (zh) * 2019-08-19 2022-05-27 喜乐医疗器材股份有限公司 止血装置和方法
CN111790004A (zh) * 2020-06-17 2020-10-20 天津市康婷生物工程集团有限公司 一种通用型载药钙磷骨水泥多孔支架制备方法

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AU2002327007B2 (en) 2005-10-20
CA2460843A1 (fr) 2003-03-27
EP1446445A4 (fr) 2007-04-04

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