WO2017203331A1 - Composition et méthodes pour l'ingénierie des tissus osseux - Google Patents

Composition et méthodes pour l'ingénierie des tissus osseux Download PDF

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WO2017203331A1
WO2017203331A1 PCT/IB2016/053147 IB2016053147W WO2017203331A1 WO 2017203331 A1 WO2017203331 A1 WO 2017203331A1 IB 2016053147 W IB2016053147 W IB 2016053147W WO 2017203331 A1 WO2017203331 A1 WO 2017203331A1
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rhbmp
bone
matrix
cells
kit
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PCT/IB2016/053147
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English (en)
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Richard MIRON
Matthias MOTTINI
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Universität Bern
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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
    • A61L27/14—Macromolecular materials
    • A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • 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
    • A61L27/225—Fibrin; Fibrinogen
    • 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
    • A61L27/24—Collagen
    • 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
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A61L27/3608—Bone, e.g. demineralised bone matrix [DBM], bone powder
    • 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
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3641—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the site of application in the body
    • A61L27/3645—Connective tissue
    • A61L27/365—Bones
    • 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
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
    • A61L27/3843—Connective tissue
    • A61L27/3847—Bones
    • 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
    • A61L27/52—Hydrogels or hydrocolloids
    • 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
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • 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
    • 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
    • A61L27/58—Materials at least partially resorbable by the body
    • 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
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
    • 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
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/12—Materials or treatment for tissue regeneration for dental implants or prostheses
    • 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
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/38—Materials or treatment for tissue regeneration for reconstruction of the spine, vertebrae or intervertebral discs

Definitions

  • the present invention relates to bone tissue engineering, in particular to
  • compositions, kits and methods for promoting bone growth and/or regeneration in patients in need thereof are provided.
  • Bone formation is a well-orchestrated process of osteoblast lineage-specific differentiation.
  • pluripotent mesenchymal stem cells differentiate into preosteoblasts rather than serving as progenitor cells for myocytes, adipocytes, or chondrocytes. These preosteoblasts then differentiate into mature osteoblasts that deposit the necessary components to form bone matrix and allow subsequent mineralization.
  • Bone morphogenetic proteins (BMPs) play an important role in regulating osteoblast differentiation and subsequent bone formation.
  • Bone defects caused by, e.g., trauma, inflammation, disease, and fracture often require graft materials for regeneration.
  • graft materials autologous bone grafts having osteoconductivity, osteoinductivity, and osteogenecity are widely used for the treatment of bone repair and regeneration.
  • the graft material For effective bone repair and regeneration, osteogenesis needs to be induced after the graft material has been implanted.
  • the graft material must not only have a three-dimensional, often porous structure, but also be able to mimicry the
  • extracellular matrix in order to produce bone tissue.
  • ECM extracellular matrix
  • the graft material is combined with growth factors, in particular BMPs.
  • BMPs growth factors
  • rh human recombinant
  • RhBMP-2 is approved for anterior lumbar interbody fusion in skeletally mature patients and rhBMP-7 received a humanitarian use device approval in 2003 for revision intertransverse lumbar fusion in
  • rhBMP-2 resulting from dosage requirements and/or side effects associated with its administration
  • various approaches have been chosen, e.g., combining the administration of rhBMP-2 with LIM Mineralization Protein (LMP-1 ) (see, e.g., US Patent Publication 20090054313, which is incorporated herein by reference in its entirety).
  • LMP-1 LIM Mineralization Protein
  • BMP-2 non-glycosylated BMP-2.
  • BMPs produced via bacterial expression systems are non-glycosylated (ng) whereas recombinant equivalents produced in mammalian cell expression systems are glycosylated (g) proteins.
  • gBMP-2 being less soluble and thus released more slowly from a bone substrate or matrix, makes it an attractive choice for reducing the amount of BMP that needs to be included with the bone substrate or matrix.
  • ngBMP-2 loaded implants indeed induced more bone formation at lower concentrations from 4-weeks onward compared to glycosylated human recombinant BMP-2 (gBMP-2) equivalents, thus indicating the value of ngBMP-2 as a potential alternative for mammalian produced recombinant BMP-2 for bone regenerative therapies (van de Watering FC, 2012).
  • BMP-2 adenoviral vectors transduced osteoblast progenitor cells with high efficiency to continuously produce biologically active BMPs inside the mammalian cells.
  • ad adenoviral vectors transduced osteoblast progenitor cells with high efficiency to continuously produce biologically active BMPs inside the mammalian cells.
  • cells transfected with adBMP-2 showed an activity of the early osteogenic marker, alkaline phosphates (ALP), that was increased by 169-fold (about 3000 nmol/min/mg) over reference cells just transfected with GDP, while cells transfected with adBMP-9 showed an activity of ALP that was increased by 273-fold (about 4800 nmol/min/mg) over reference cells just transfected with GDP.
  • ALP alkaline phosphates
  • the adBMP-9 mediated activity was about 1 .6 higher than the adBMP-2 mediated activity (Kang et al. 2004).
  • Human BMP-9 also known as growth and differentiation factor 2 (GDF-2), is a member of the BMP subgroup of the TGF-beta superfamily proteins that signal through heterodimeric complexes composed of type I and type II BMP receptors.
  • BMP-9 regulates the development and function of a variety of embryonal and adult tissues.
  • a human BMP-9 cDNA may encode a 429 amino acid (aa) precursor that includes a 22 aa signal sequence, a 298 aa propeptide, and a 1 1 1 aa mature protein. Unlike with other BMP family proteins, the propeptide does not interfere with the biological activity of BMP-9 and remains associated with the mature peptide after proteolytic cleavage.
  • human BMP-9 (SEQ ID NO: 1 ) shares 64% aa sequence identity with human BMP- 10 and less than 50% aa sequence identity with other BMPs.
  • BMP-9 is expressed by non-parenchymal cells in the liver, where it promotes lipid metabolism and inhibits glucose production. BMP-9 exerts a prolonged hypoglycemic effect which may be due to an enhancement of insulin release.
  • BMP-9 interacts with a high affinity specific heteromeric receptor expressed on liver endothelial cells that has been identified as ALK-1 .
  • BMP-9 functions in the development and maintenance of the cholinergic neuronal phenotype.
  • BMP-9 also induces the differentiation of mesenchymal stem cells into the chondrogenic lineage. At low concentrations, BMP- 9 is a proliferative factor for hematopoietic progenitor cells, but at higher
  • a BMP preferably a rhBMP
  • a rhBMP that can be administered at doses lower than rh-BMP-2 to limit secondary side effects.
  • a rhBMP that has an acceptable biological half-life and which is able to mimic the route of administration in vivo of a BMP-producing cell.
  • the present invention addresses one or more of those needs and/or other needs in the art.
  • the present invention is, in one embodiment, directed to a method for promoting bone growth and/or regeneration comprising:
  • administering to a patient, in particular a human patient in need thereof:
  • rhBMP-9 at a concentration of less than 1 .5 mg/ml, less than 1 mg/ml, less than 0.5 mg/ml, less than 0.2 mg/ml or less than 0.1 mg/ml, wherein the rhBMP-9 is optionally glycosylated rhBMP-9 (gBMP-9).
  • the substrate may be an implant such as a dental implant, a surgical implant including a plastic surgical implant or an orthopedic implant.
  • the substrate may comprise ceramics, titanium, collagen and zirconium.
  • the rhBMP-9 may coat at least a part of said substrate or matrix and/or may be applied intra-osseal.
  • the promoting bone growth and/or regeneration may comprise differentiation into osteoblasts, proliferation of osteoblasts and/or adhesion of osteoblasts to the substrate or matrix.
  • a patient of the method may suffer from osteoporosis or may have suffered bone loss, such as loss at the, e.g., jaw bone.
  • the method may comprise administering (a) and (b) as part of a bone tissue, wherein the bone tissue comprises cells selected from the group consisting of osteogenic cells, pluripotent stem cells, mesenchymal cells, and embryonic stem cells to induce bone formation.
  • the bone tissue comprises cells selected from the group consisting of osteogenic cells, pluripotent stem cells, mesenchymal cells, and embryonic stem cells to induce bone formation.
  • the present invention is also directed to a composition or kit for promoting bone growth and/or inhibiting bone loss.
  • composition or kit may comprise
  • rhBMP-9 comprises amino acids 318 to 429 or 320 to 429 of SEQ ID NO: 1 or has more than 80%, 90%, 95%, 96%, 97%, 98%, 99% and/or complete sequence identify with amino acids 318 to 429 or 320 to 429 of SEQ ID NO: 1 , and/or
  • rhBMP-9 comprises at least 80, 90, 100 consecutive amino acids of amino acids 318 to 429 or 320 to 429 of SEQ ID NO: 1 ; or
  • kits generally contains (a) and (b), which are generally provided in separate (at least two, in certain embodiment three or more) containers and generally include instructions for using (a) and (b) together for promoting said bone growth and/or inhibiting said bone loss.
  • the one or more substrates or matrixes may comprise or constitute a membrane or a sponge.
  • the one or more substrates or matrixes may be made of one or more of the following materials: autogenous bone; an allograft such as, optionally demineralized, freeze- dried bone allograft or an allograft bone block; xenografts such as porcine bone grafts, bovine bone grafts, mineralized bovine bone, a deproteinized bovine derived bone mineral, a natural bovine derived bone mineral or a combination thereof.
  • an allograft such as, optionally demineralized, freeze- dried bone allograft or an allograft bone block
  • xenografts such as porcine bone grafts, bovine bone grafts, mineralized bovine bone, a deproteinized bovine derived bone mineral, a natural bovine derived bone mineral or a combination thereof.
  • the at least one liquid matrix may comprise a fibrin, fibrinogen, aprotinin, factor XIII, thrombin, calcium chloride or a combination thereof, optionally in two separate containers, and/or at least one gel matrix, such as a nanogel matrix or a hydrogel matrix such as a hyaluronic acid matrix.
  • the one or more substrates or matrixes may comprise cross-linked or non-cross-linked collagen, collagen bi-products comprising, e.g., synthetic or natural bone material, e-PTFE, d-PTFE, or combinations thereof.
  • the one or more substrates or matrixes may comprise cross-linked or non-cross- linked collagen and one or more bone graft materials, wherein said collagen preferably covers said bone graft material.
  • the one or more substrates or matrixes may comprise calcium phosphates, calcium sulfurs, hydroxyapatite and derivatives thereof, biphasic calcium phosphates, orthophosphates, beta-tricalcium phosphates, alpha-tricalcium phosphates or combinations thereof.
  • the one or more substrates or matrixes may comprise polyesters such as poly(glycolic acid) (PGA), poly(lactic acid) (PLA), poly ( ⁇ -caprolactone) (PCL), or co-polymers and/or combinations thereof.
  • composition or kit may further comprise further growth factors, such as human recombinant growth factors, such as BMP1 , BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8, BMP10, BMP1 1 , BMP12, BMP13 BMP14, BMP15, BMP16, GDF1 , GDF3, GD8, GDF9, GDF12, GDF14, PDGF, IGF, EGF, FGF2, FGF19 and mixtures thereof.
  • the rhBMP-9 or the amino acid sequence may cover the substrate or matrix, wherein the matrix and/or the rhBMP-9 or part thereof may be applied via 3-D printing technologies.
  • the rhBMP-9 or the amino acid sequence may be part of any one of the compositions or kits disclosed herein.
  • the present invention is also directed to a method for promoting bone growth and/or regeneration comprising administering to a human patient in need thereof any of the compositions, kits or any component or combination of components of the composititon described herein.
  • the present invention is also directed to the use of any of the compositions, kits or any component or combination of components of the compositions described herein, preferably for promoting bone growth and/or regeneration, e.g., in a live mammal such as a human.
  • Figure 1 shows SEM (scanning electron microscope) images of collagen
  • Figure 2 shows an attachment assay of cells of the stromal cell line ST2, derived from mouse bone marrow (pluripotent mesenchymal) seeded on 1 ) a control, 2) rhBMP-2 low (10 ng/ml), 3) rhBMP-2 high (100 ng/ml), 4) rhBMP-9 low (10 ng/ml) and 5) rhBMP-9 high (100 ng/ml), with membranes at 8 hours post seeding. It can be seen that no significant differences were observed between the groups. All references to "BMP" in the figures 2-33 stand for rhBMPs produced in the CHO cells specified in the Material and Methods section, reagents and cell lines.
  • Figure 3 shows the results a proliferation assay of ST2 cells seeded on 1 ) a control, 2) rhBMP-2 low (10 ng/ml), 3) rhBMP-2 high (100 ng/ml), 4) rhBMP-9 low (10 ng/ml) and 5) rhBMP-9 high (100 ng/ml), with membranes at 1 , 3 and 5 days post seeding. It can be seen that only rhBMP-9 demonstrated significantly lower levels when compared to control samples at 3 days ( * denotes significant difference, p ⁇ 0.05).
  • Figure 4 shows data obtained from a real-time PCR of ST2 cells seeded on 1 ) a control, 2) rhBMP-2 low (10 ng/ml), 3) rhBMP-2 high (100 ng/ml), 4) rhBMP-9 low (10 ng/ml) and 5) rhBMP-9 high (100 ng/ml), with membranes for genes encoding (A) Runx2, (B) alkaline phosphatase (ALP), (C) bone sialoprotein (BSP) at 3 and 14 days post seeding ( * denotes significant difference, p ⁇ 0.05; # denotes control samples significantly lower than all other modalities, p ⁇ 0.05; ** denotes significantly higher than all other treatment modalities, p ⁇ 0.05).
  • A Runx2
  • ALP alkaline phosphatase
  • BSP bone sialoprotein
  • Figure 5 is a visual representation of alizarin red stained particles on A) a control, B) rhBMP2 low (10 ng/ml), C) rhBMP2 high (100 ng/ml), D) rhBMP9 low (10 ng/ml) and E) rhBMP9 high (100 ng/ml), with membranes at 14 days post seeding. The intensity of dark staining on membrane with rhBMP9 in comparison to control and rhBMP2 samples is notable. F) Quantified data of alizarin red staining from color thresholding software for ST2 cells ( * denotes significant difference, p ⁇ 0.05).
  • Figure 6 is a visual representation of alizarin red staining (in vitro mineralization) at 14 days for bone forming osteoblasts seeded at 5 concentrations of rhBMPs including 1 ) a control, 2) 10ng/ml 3) 50ng/ml, 4) 100ng/ml and 5) 200ng/ml for each of recombinant rhBMP-2, 7 and 9.
  • B Quantified data of alizarin red staining from color thresholding software for ST2 cells ( * denotes significant difference between rhBMP-9 samples and both rhBMP-2 or rhBMP-7, p ⁇ 0.05, "denotes significantly higher than all other treatment modalities, p ⁇ 0.05).
  • Figure 7 shows alkaline phosphatase staining of NBM particles at 7 days post seeding. Web-like structures were found on NBM particles previously coated with rhBMP-9 either at low or high dose (see, e.g. the description of Fig. 5 for what constitutes a low and high dose).
  • Figure 8 shows alkaline phosphatase staining of ST2 cells seeded on 1 ) a control, 2) rhBMP-2 low (10 ng/ml), 3) rhBMP-2 high (100 ng/ml), 4) rhBMP-9 low (10 ng/ml) and 5) rhBMP-9 high (100 ng/ml), NBM bone grafts at 7 days post seeding.
  • RhBMP- 9 low and high significantly increased ALP staining when compared to control and rhBMP-2 samples ( ** denotes significantly higher than all other treatment modalities, p ⁇ 0.05).
  • Figure 9 shows the results of real-time PCR of ST2 cells seeded on 1 ) a control, 2) rhBMP-2 low (10 ng/ml), 3) rhBMP-2 high (100 ng/ml), 4) rhBMP-9 low (10 ng/ml) and 5) rhBMP-9 high (100 ng/ml), NBM bone grafts for genes encoding (A) Runx2, (B) Collagen 1 alpha 2 (COL1 a2), (C) alkaline phosphatase (ALP) and (D) osteocalcin (OCN) at 3 and 14 days post seeding ( * denotes significant difference, p ⁇ 0.05; # denotes control samples significantly lower than all other modalities, p ⁇ 0.05; ** denotes significantly higher than all other treatment modalities, p ⁇ 0.05).
  • Figure 10 is a visual representation of alizarin red stained particles on 1 ) a control, 2) rhBMP-2 low (10 ng/ml), 3) rhBMP-2 high (100 ng/ml), 4) rhBMP-9 low (10 ng/ml) and 5) rhBMP-9 high (100 ng/ml), NBM bone grafts at 14 days post seeding. Note the intensity of staining on particles coated with rhBMP-9 in comparison to control and rhBMP-2 samples.
  • Figure 11 shows in (A) an attachment assay of ST2 cells seeded on (i) TISSEEL only, (ii) TISSEEL + rhBMP-9 (100 ng/ml) at 8 hrs. No significant differences were observed between the 2 groups.
  • Figure 12 shows the alkaline phosphatase (ALP) staining of ST2 cells treated on (i) control plastic dishes, (ii) TISSEEL, and (iii) TISSEEL + rhBMP-9 (100 ng/ml) at 7 days post-seeding.
  • A It was visually observed that rhBMP-9 induced ALP staining when compared either with control plastic or TISSEEL only samples.
  • B Quantified data of ALP staining from color thresholding software, ("denotes significantly higher than all other treatment modalities, p ⁇ 0.05difference, P ⁇ 0.05). TISSEEL combined with rhBMP-9 significantly induced higher ALP staining when compared to control plastic and TISSEEL only samples. The assays were performed in triplicate.
  • Figure 13 shows the results of real-time PCR of ST2 cells seeded on (i) a control (Plastic), (ii) TISSEEL only, and (iii) TISSEEL + rhBMP-9 (100 ng/ml) for genes encoding (A) Runx2, (B) alkaline phosphatase (ALP), (C) bone sialoprotein (BSP) and (D) osteocalcin (OCN) at 3 and 14 days post seeding ( ** denotes significantly higher than all other treatment modalities, p ⁇ 0.05).
  • TISSEEL combined with rhBMP- 9 demonstrated significantly higher ALP mRNA levels (2 fold) at 3 days when compared to control and TISSEEL only.
  • TISSEEL + rhBMP-9 significantly induced BSP levels 8 fold at 14 days, and also OCN levels 6 fold at 3 days and 4 fold at 14 days when compared to control plastic and TISSEEL only samples. The assays were run in triplicate.
  • Figure 14 provides in (A) a visual representation of alizarin red stained particles on (i) control (plastic), (ii) TISSEEL only, and (iii) TISSEEL + rhBMP-9 (100 ng/ml) at 14 days post seeding. Red staining calcified nodules were widely observed in TISSEEL + rhBMP-9 samples.
  • Figure 15 shows in (A) an attachment assay of ST2 cells seeded on (i) HA only, (ii) HA + BMP9 (100 ng/ml) at 8 hrs. HA coated with rhBMP-9 significantly induced higher cell adhesion when compared to HA only at 8 hrs post cell seeding. (B) Proliferation assay of ST2 cells seeded on (i) a control (Plastic dish), (ii) HA only, (iii) HA+ rhBMP-9 (100 ng/ml) at 1 , 3 and 5 days post-seeding.
  • Figure 16 shows alkaline phosphatase (ALP) staining of ST2 cells treated on (i) control plastic dishes, with (ii) HA, and (iii) HA + rhBMP-9 (100 ng/ml) at 7 days post- seeding.
  • A It was visually observed that HA combined with rhBMP-9 induced ALP staining when compared to either control tissue-culture plastic or HA only samples.
  • B Quantified data of ALP staining from color thresholding software, ("denotes significantly higher than all other treatment modalities, p ⁇ 0.05). HA combined with rhBMP-9 significantly promoted ALP staining when compared to control plastic and HA only samples. The assays were performed in triplicate.
  • Figure 17 provides in (A) a visual representation of alizarin red staining on (i) HA (without cells) (ii) without HA (plastic), (iii) HA only, and (iv) HA + rhBMP-9 (100 ng/ml) at 14 days post seeding. Red stained-calcified nodules were widely observed in the HA + rhBMP-9 group.
  • HA demonstrated significantly higher Alizarin red staining when compared to control plastic dish samples.
  • HA + rhBMP-9 significantly increased Alizarin red staining when compared with the other modarlities. The assays were performed in triplicate.
  • Figure 18 shows in (A) an attachment assay of ST2 cells seeded on (i) a control (collacone only), (ii) collacone + rhBMP-2 low (10 ng/ml), (iii) collacone + rhBMP-2 high (100 ng/ml), (iv) collacone + rhBMP-9 low (10 ng/ml) and (v) collacone + rhBMP-9 high (100 ng/ml) at 8 hrs. No significant differences were observed between 5 groups at 8 hrs post cell seeding.
  • Figure 19 shows the results of alkaline phosphatase (ALP) staining of ST2 cells treated on (i) a control (Plastic dishes), (ii) collacone only, (iii) collacone + rhBMP-2 low (10 ng/ml) (iv) collacone + rhBMP-2 high (100 ng/ml), (v) collacone + rhBMP-9 low (10 ng/ml) (vi) collacone + rhBMP-9 high (100 ng/ml) at 7 days post-seeding. (A) It was visually observed that rhBMP-9 induced ALP staining when compared with all other groups.
  • ALP alkaline phosphatase
  • Figure 20 shows in (A) a visual representation of alizarin red staining on (i) collacone (without cells) (ii) without collacone (plastic, with cells), (iii) collacone only, (iv) collacone + rhBMP-2 low (10 ng/ml), (v) collacone + rhBMP-2 high (100 ng/ml), (vi) collacone + rhBMP-9 low (10 ng/ml), ad (vii) collacone + rhBMP-9 high (100 ng/ml) at 14 days post cell seeding. Red calcified nodules were widely observed in collacone + rhBMP-9 group.
  • Figure 21 shows in (A) an attachment assay of ST2 cells seeded on either Bio-Oss® or Hypro-Oss® with (i) a control (graft only), (ii) rhBMP-2 low (10 ng/ml), (iii) rhBMP- 2 high (100 ng/ml), (iv) rhBMP-9 low (10 ng/ml) and (v) rhBMP-9 high (100 ng/ml) at 8 hrs post cells seeding.
  • Figure 22 shows the results of alkaline phosphatase (ALP) staining of ST2 cells treated at 7 days post-seeding.
  • ALP staining of Bio-Oss® particles at 7 days post-seeding with i) Bio-Oss® only, (iii) rhBMP-2 low (10 ng/ml) (iv) rhBMP-2 high (100 ng/ml), (v) rhBMP-9 low (10 ng/ml) (vi) rhBMP-9 high (100 ng/ml).
  • C Quantified data of ALP staining from color thresholding software.
  • Bio-Oss® coated with rhBMP-9 high group demonstrated significantly higher ALP staining when compared to control groups.
  • ALP activities on Hypro-Oss® coated with rhBMP-9 either at low or high dose were significantly induced at 7 days post cell seeding when compared with the other modalities.
  • the assays were performed in triplicate.
  • Figure 23 is a visual representation of alizarin red-stained particles on (i) a control, (ii) rhBMP-2 low (10 ng/ml), (iii) rhBMP-2 high (100 ng/ml), (iv) rhBMP-9 low (10 ng/ml) and (v) BMP-9 high (100 ng/ml), either (A) Bio-Oss® or (B) Hypro-Oss® at 14 days post-seeding. Note the intensity of staining on particles coated with BMP-9 in comparison with control and rhBMP-2 samples.
  • Figure 24 shows in (A) an attachment assay of ST2 cells seeded either on DBX or MAXRESORB with (i) a control (bone graft only), (ii) rhBMP-2 low (10 ng/ml), (iii) rhBMP-2 high (100 ng/ml), (iv) rhBMP-9 low (10 ng/ml) and (v) rhBMP-9 high (100 ng/ml) at 8 hrs post-seeding. It was observed BMP-2 high dose samples
  • Figure 25 shows the results of alkaline phosphatase (ALP) staining of ST2 cells treated at 7 days post-seeding.
  • ALP staining of DBX particles at 7 days post- seeding with i) DBX only, (iii) rhBMP-2 low (10 ng/ml) (iv) rhBMP-2 high (100 ng/ml),
  • rhBMP-9 high 100 ng/ml. Higher ALP staining was observed either on DBX or MAXRESORB particles previously coated with rhBMP-9 either at low or high dose.
  • C, D Quantified data of ALP staining on (C) DBX, (D) MAXRESORB from color thresholding software. ( * denotes significant difference, p ⁇ 0.05, ** denotes significantly higher than all other treatment modalities, p ⁇ 0.05).
  • DBX precoated with high dose of rhBMP-9 demonstrated significantly higher ALP staining when compared to control samples.
  • MAXRESORB precoated with rhBMP-9 either at low or high dose significantly induced ALP activities when compared with the other modalities. The assays were performed in triplicate.
  • Figure 26 shows a visual representation of alizarin red-stained particles on (i) control, (ii) rhBMP-2 low (10 ng/ml), (iii) rhBMP-2 high (100 ng/ml), (iv) rhBMP-9 low (10 ng/ml) and (v) BMP-9 high (100 ng/ml), either (A) DBX or (B) MAXRESORB at 14 days post-seeding. Note the intensity of staining on particles coated with BMP-9 in comparison with control and rhBMP-2 samples. (C, D) Quantified data of alizarin red staining from color thresholding software ( * denotes significant difference, p ⁇ 0.05).
  • Rh BMP-2 high and rhBMP-9 high groups significantly increased Alizarin red staining on DBX when compared with control plastic dishes
  • rhBMP-2 precoated MAXRESORB demonstrated significantly higher alizarin red staining when compared with control groups
  • rhBMP-9 precoated MAXRESORB demonstrated significantly further higher alizarin red staining when compared with rhBMP-2 samples.
  • the assays were performed in triplicate.
  • Figure 27 shows in (A) an attachment assay of ST2 cells seeded on (i) a control (MAXRESORB inject only), and (ii) MAXRESORB inject + rhBMP-9 (100 ng/ml) at 8 hrs poste seeding. No significant differences were observed between 2 groups. (B) Proliferation assay of ST2 cells seeded on (i) a control (Plastic dish), (ii)
  • MAXRESORB inject only (iii) MAXRESORB inject+ rhBMP-9 (100 ng/ml) at 1 , 3 and 5 days post-seeding.
  • MAXRESORB groups significantly decreased cell numbers when compared to on plastic dishes at 1 , 3 and 5 days post seeding.
  • MAXRESORB inject + rhBMP-9 demonstrated higher cell proliferation when compared to
  • MAXRESORB inject control samples at 3 days post cell seeding. ( * denotes significant difference, p ⁇ 0.05, "denotes significantly higher than all other modalities, p ⁇ 0.05). The assays were run in triplicate.
  • Figure 28 shows alkaline phosphatase (ALP) staining of ST2 cells treated on (i) control plastic dishes, (ii) MAXRESORB inject only and (iii) MAXRESORB inject + rhBMP-9 (100 ng/ml) at 7 days post-seeding. (A) It is visually observed on
  • MAXRESORB inject that rhBMP-9 induced ALP staining when compared with MAXRESORB inject only.
  • B Quantified data of ALP staining from color thresholding software. ( ** denotes significantly higher than all other treatment modalities, p ⁇ 0.05). rhBMP-9 precoated MAXRESORB inject demonstrated significantly higher ALP staining when compared to control plastic and MAXRESORB inject alone. The assays were performed in triplicate.
  • Figure 29 shows in (A) a visual representation of alizarin red stained particles on (i) MAXRESORB block only, (ii) MAXRESORB block + rhBMP-9 high (100 ng/ml) at 14 days post cell seeding. Note the intensity of staining on blocks coated with rhBMP-9 in comparison with control samples. (B) Quantified data of alizarin red staining from color thresholding software ( * denotes significant difference, p ⁇ 0.05). MAXRESORB block coated with rhBMP-9 demonstrated significantly higher Alizarin red staining when compared to control samples. The assays were performed in triplicate.
  • Figure 30 shows in (A) an attachment assay of ST2 cells seeded on (i) Jason only, (ii) Jason + rhBMP-2 (100 ng/ml), (iii) Jason + rhBMP-9 (100 ng/ml), (iv)
  • MUCODERM only, (v) MUCODERM + rhBMP-2 (100 ng/ml), (vii) MUCODERM + rhBMP-9 (100 ng/ml) at 8 hrs.
  • rhBMP-2-precoated Jason membrane significantly induced cell adhesions when compared to the other modalities at 8 hrs post cell seeding, whereas no significant differences were observed between MUCODERM membrane samples.
  • Figure 31 shows an alkaline phosphatase (ALP) staining of ST2 cells treated either on (A) JASON or (B) MUCODERM membrane with (i) membrane only, (ii) rhBMP-2 (100 ng/ml), (iii) rhBMP-9 high (100 ng/ml) at 7 days post-seeding. It is visually observed that rhBMP-9 induced ALP staining when compared with membrane only.
  • C,D Quantified data of ALP staining from color thresholding software. ( * denotes significant difference, p ⁇ 0.05, "denotes significantly higher than all other treatment modalities, p ⁇ 0.05).
  • RhBMP-9 precoated Jason membrane demonstrated significantly higher ALP activities when compared to the other modalities (C).
  • RhBMP-9 on MUCODERM demonstrated significantly higher ALP activities when compared with control samples (D).
  • the assays were performed in triplicate.
  • Figure 32 is a visual representation of alizarin red staining on (A) JASON or (C) MUCODERM membrane with (i) membrane only, (ii) rhBMP-2 (100 ng/ml), (iii) rhBMP-9 high (100 ng/ml) at 14 days post-seeding. Note the intensity of staining on membranes coated with rhBMP-9 in comparison with control and rhBMP-2 samples.
  • B, D Quantified data of alizarin red staining from color thresholding software ( * denotes significant difference, p ⁇ 0.05).
  • C RhBMP-9 precoated JASON
  • Figure 33 shows in (A) round-shaped full thickness bone defects from a 6 mm diameter trephine drill in rabbit parietal bone: (i) NC (negative control, empty), (ii) BO (Bio-Oss®) (iii) BO + rhBMP-2_5 g (iv) BO + rhBMP-2_20 g (v) BO + rhBMP- 9_5 ⁇ g, and (vi) BO + rhBMP-9_20 ⁇ g were applied into bone defects and covered with BioGide®.
  • C New bone volume (BV) was measured at bone defect area. Bio-Oss® coated either with rhBMP-9_5 ⁇ g or rhBMP-9_20 ⁇ g specimens induced significantly newly formed bone when compared with BO alone group. ( * denotes significant difference, p ⁇ 0.05; # denotes significantly lower than all other modalities, p ⁇ 0.05).
  • a substrate according to the present invention has a structural function upon implantation into the body of a patient, replacing part of a solid bone of said patient. For example, it might replace a part of a larger bone such as the jaw bone.
  • the substrate is an implant.
  • a matrix according to the present invention carries the rhBMP-9 described herein.
  • a substrate might or might not be a matrix according to the present invention.
  • a substrate is a matrix if the rhBMP-9 is directly applied onto the substrate.
  • a matrix might be applied to a substrate and in turn carry the rhBMP-9.
  • the bone graft is the substrate and the collagen is the matrix.
  • the bone graft is both substrate and matrix.
  • the substrate and/or matrix according to the present invention is three-dimensional, might be made out of non-resorbable or resorbable material, might be structured, e.g. as a scaffold that maximizes surface volume or might be unstructured and take, e.g., the form of a simple membrane.
  • a substrate or matrix might be an autogenous bone; an allograft such as a (demineralized) freeze-dried bone allograft (e.g., DFDBA (demineralized) or DBX from human) or an allograft bone block; a xenograft such as porcine bone, bovine bone including mineralized bovine bone; a synthetic bone graft, e.g. from hydroxyapatite (MAXRESORB, BOTISS) or a combination thereof.
  • an allograft such as a (demineralized) freeze-dried bone allograft (e.g., DFDBA (demineralized) or DBX from human) or an
  • a substrate might also be an implant, such as a dental implant, a surgical implant including a plastic surgical implant or an orthopedic implant, which may be made of or comprise ceramics, titanium, collagen and/or zirconium.
  • Matrixes may take the form of minerals such as deproteinized bovine derived bone mineral (BioOssTM) or a natural bovine derived bone mineral (e.g.,Hypro-OssTM).
  • a matrix might also be a liquid matrix comprising fibrinogen, fibrin, aprotinin, factor XIII, thrombin, calcium chloride or a combination thereof.
  • a matrix might also be a gel matrix such as a nanogel matrix or a hydrogel matrix, such as a hyaluronic acid matrix, e.g., obtained by REGEDENT (see, Material and Methods).
  • the liquid matrix comprising fibrogen is a fibrin sealant (FS).
  • FSs have many surgical applications and comprise next to fibrogen (factor I (Fl)), primarily Flla, calcium chloride, and, occasionally, activated Factor XIII (FXIIIa).
  • Factor I Factor I
  • the FSs are often provided as two component tissue adhesive systems mimicking natural clot cascade, one component being a protein solution containing fibrinogen as the main active ingredient, the other component being a thrombin solution comprising thrombin, e.g., human thrombin, as the main active ingredient.
  • the two components of the fibrin sealant may be stored as a sterilized lyophilized powder.
  • components may be reconstituted into liquid form by adding distilled water. As the two components are mixed together, they generally polymerize at the site of application into a relatively dense gel. Thrombin in combination of Ca 2+ may catalyze polymerization of the fibrinogen, converting the fibrinogen into fibril polymer. Further, thrombin and Ca 2+ may activate coagulation factor XII I, which leads to covalent crosslinking of fibrin. The rate of proteolytic degradation of the fibrin polymer clot may be decreased and mechanical stability may be increased as a result of the covalent crosslinking of the polymer.
  • the (i) thrombin solution and (ii) protein solution might be kept separate, e.g., each in a chamber of one double-chamber syringe.
  • the active ingredients may be fractionated from pooled human plasma.
  • the active ingredients preferably comprise in addition to fibrinogen, aprotinin (e.g., synthetic), factor XIII, or combinations thereof, e.g., in the protein solution.
  • the active ingredients may also comprise in addition to thrombin (e.g., human) calcium chloride (e.g., 2 H2O), e.g., in the thrombin solution.
  • the rhBMP-9 may be added to or be part of either of the solutions (thrombin/protein) prior to mixing them together.
  • a preferred FS is, e.g., commercially available under the trademark TISSEEL
  • FSs distributed by Baxter.
  • Other commercially available FSs such as BERIPLAST (Behringwerke AG), TISSUCOL (Baxter), and BIOCOL (CRTS) are also part of the present invention.
  • a substrate or matrix may comprise or be made of cross-linked or non-cross-linked collagen in form of e.g. sponges or membranes, collagen bi-products comprising synthetic or natural bone material (e.g., HYPRO-OSS), e-PTFE, d-PTFE, or combinations thereof.
  • Cross-linked or non-cross-linked collagen might cover of printed on bone graft material (suitable materials are also disclosed in U.S. Pat. Nos. 6,919,308; 6,949,251 ; and 7,041 ,641 which are incorporated herein by reference).
  • the substrate or matrix may comprise calcium phosphates, calcium sulfurs, hydroxyapatite and derivatives thereof, biphasic calcium phosphates,
  • the substrate or matrix may in addition or alternatively comprise polyesters such as poly(glycolic acid) (PGA), poly(lactic acid) (PLA), poly ( ⁇ -caprolactone) (PCL), or co-polymers and/or combinations thereof.
  • the rhBMP-9 optionally including the matrix, is in certain embodiments applied onto the substrate or matrix via 3-D printing technologies and covers the substrate or matrix, preferably as a three-dimensional rhBMP-9 pattern.
  • a 3D printing technology refers to a manufacturing method that uses a layer-by-layer process to build objects.
  • 3D printing technology can, e.g., dispense growth factors, and even live cells with hydrogel, at the desired position until the 3D tissue is built up. Therefore, 3D printing can produce structures that have the spatial features of the native tissue.
  • the material to be printed may, e.g., be introduced into a plastic syringe equipped with tapered plastic nozzle. Pneumatic pressure can be applied to dispense the material.
  • a print pattern may be produced by moving the printing nozzle at a certain speed.
  • rhBMP-9 can and was in fact loaded onto different matrixes/substrates, namely 1 ) fibrin sealants (e.g., TISSEEL), 2) hyaluronic acid gel carrier systems (e.g., one by Regedent), 3) collagen sponges as such utilized during routine extraction socket healing (COLLACONE by Botiss), 4) bovine derived minerals that may include collagen such as Hypro-Oss® (Biolmplon), 5)
  • deproteinized bovine derived bone minerals such as BioOss® (Geistlich), 6) demineralized freeze-dried allograft, e.g., from human origin such as DBX
  • rhBMP-9 may be loaded onto the matrix/substrate.
  • the matrix/substrate is immersed in an rhBMP-9 solution, for example the
  • matrix/substrate is immersed at 35-39 ° C in, e.g., a suitable volume of solution containing 10ng-150 mg/ml rhBMP-9, such as 10ng-100mg/ml, 10ng-80mg/ml, 10ng- 60mg/ml, 10ng-40mg/ml, 10ng-20mg/ml, 10ng-10mg/ml, 10ng-5mg/ml, 10ng- 2mg/ml, 20ng-100mg/ml, 20ng-80mg/ml, 20ng-60mg/ml, 20ng-40mg/ml, 20ng- 20mg/ml, 20ng-10mg/ml, 20ng-5mg/ml, 20ng-2mg/ml rhBMP-9 for at least 10min, 20min, 30min, 45min and up to 60min,2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 18, 24 hours, 36 hours, 48 hours .
  • HA hydroxyapatite
  • materials that undergo conversions also allow for incorporation during this conversion (e.g., dicalcium phosphate dihydrate (DCPD) conversion to HA).
  • DCPD dicalcium phosphate dihydrate
  • the highest rhBMP-9 uptake can generally be achieved by using the immersion method. However, it is generally preferred to use the incorporation method (which may or may not be the immersion method) that allows for the most consistent and longest release of rhBMP-9 over time (sustained and prolonged release, respectively). Thus, in certain cases, such as in the case of HA, incorporation by precipitation may be preferred.
  • the amount of rhBM-9 incorporated can be determined by subtracting the rhBMP concentration remaining in, e.g., the immersion solution at the end of the loading process (e.g., before filtration) from the initial rhBMP concentration in the solution.
  • the rate of rhBMP-9 release can be determined by incubating the rhBMP-loaded substrate/matrix (and a control without rhBMP-9) in, e.g., PBS.
  • Supernatants are withdrawn and optionally frozen, e.g., at 12 hours and at days 1 , 3, 7, and 14. The removed supernatant may be replaced with an equal amount of, e.g., fresh PBS at each time point.
  • the protein concentration can be measured using, e.g., two different protein assays, such as, e.g., the Lavapep total protein fluorescence assay
  • rhBMP-9 enzyme-linked immunosorbent assay can be used to determine total protein uptake into the matrix/substrate and its release profile in vitro.
  • ELISA enzyme-linked immunosorbent assay
  • 50-100%, preferably more than 60%, 70%, 80% or 90% of the rhBMP-9 provided are incorporated.
  • rhBMP-9 is released for at least 5, 6, 7, 10, 14 or 21 days showing a total release rate over said time of at least 10%, 15%, 20%, 25% or more.
  • a total release rate over said time of at least 10%, 15%, 20%, 25% or more.
  • there might be one, two or more burst releases during that time e.g., after 12 hours and, e.g., 7 days.
  • rhBMP9 induced osteoblast differentiation was assessed by alkaline phosphatase (ALP) expression, alizarin red staining and/or quantifying the mRNA expression of relevant genes ("parameters for osteoblast differentiation").
  • ALP alkaline phosphatase
  • the parameters for osteoblast differentiation have, after administration using rhBMP-9, in particular gBMP-9, values that were higher than the values for the respective controls without any rhBMP, e.g., more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80 or 100 times higher.
  • the parameters for osteoblast differentiation in particular ALP staining showed absolute values over 150%, over 200%, over 300%.
  • administration of the equivalent amount of rhBMP-2 and rhBMP-9 lead, when rhBMP-9 was involved, to values for parameters for osteoblast differentiation that were more than 1 .6, 1 .8, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 times the values measured when rhBMP-2 was involved and were well beyond what could have been expected in view of the gene-therapy models previously tested.
  • Figures 1 1 -14 show the results with TISSEEL as a matrix. RhBMP-9 at
  • osteoblast alkaline phosphatase expression significantly induced osteoblast alkaline phosphatase expression, alizarin red staining and real-time PCR for genes encoding ALP, bone sialoprotein and osteocalcin.
  • Figs . 15 -17 show that a combination of hyaluronic acid (HA) as a matrix for rhBMP- 9 significantly induced alkaline phosphatase expression and alizarin red staining when compared to control samples and HA alone.
  • HA hyaluronic acid
  • Figs . 18 -20 show that the combination of COLLACONE as a matrix for rhBMP-9 significantly induced alkaline phosphatase expression and alizarin red staining when compared to control samples as well as when COLLACONE was loaded with rhBMP-2.
  • Figs . 21 -23 show that Hypro-Oss® and Bio-Oss® induced osteoblast differentiation when combined with rhBMP-9.
  • Hypro-Oss® carrying collagen furthermore significantly improved growth factor induction of osteoblast differentiation relative to Bio-Oss®.
  • Figs . 24 -26 show that both DBX and MAXRESORB synthetic bone grafts were able to stimulate in combination with rhBMP-9 new bone formation relative to control samples.
  • rhBMP-9 induced higher osteoblast differentiation relative to rhBMP-2.
  • Figs. 27- 29 show that MAXRESORB injection and MAXRESORB block combined with rhBMP-9 were both able to stimulate osteoblast differentiation as assessed by ALP activity and alizarin red staining.
  • Figs. 30-32 show that both types of collagen membranes were able to significantly induce osteoblast differentiation.
  • JASON membranes seemed to generate better results for rhBMP-9 when compared to rhBMP-2.
  • SEQ ID NO: 1 is the 429 amino acid (aa) precursor encoded by the human BMP-9 cDNA that includes a 22 aa signal sequence (underlined), a 298 aa propeptide, and a 1 1 1 aa mature protein (bold).
  • the 1 1 1 aa mature protein may be administered or the full 429 amino acid precursor (see also UniProtKB/Swiss-Prot: Q9UK05.1 , as of March 16, 2016).
  • the propeptide does not interfere with the biological activity of BMP-9 and generally remains associated with the mature peptide after proteolytic cleavage.
  • the mature peptide associated with the propeptide may also be administered in accordance to the present invention.
  • rhBMP-9 administered is preferably produced by a recombinant eukaryotic cell as further described below.
  • the rhBMP-9 may differ from the naturally occurring BMP-9 in at least one, two, three, four or more amino acids.
  • a therapeutically effective dose of rhBMP-9 is less than the currently acceptable therapeutically effective amount for rhBMP-2.
  • 1 .5 mg/ml of bone formed is the therapeutic concentration of rhBMP-2 in primates in vivo with smaller doses effective in cell culture and rodents.
  • the effective dose of rhBMP- 9 according to the present invention is less than 1 .5 mg/ml of bone formed, less than 1 .Omg/ml, less than 0.5 mg/ml, less than 0.2 mg/ml or 0.1 mg/ml, preferably at least 10-fold less than the dose required in rhBMP-2 therapy.
  • the dose required in rhBMP-2 therapy can be 20 mg rhBMP-2 per site of 10 cc of bone formation.
  • the therapeutically effective dose of rhBMP-9 is at least 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-foldless than the dose required in rhBMP-2 therapy.
  • the rhBMP-9 of the present invention may have preferably a half-life of more than 20 min., more than 30 min., more than 40 min., more than 50 min, more than 1 hour, more than 1 .5 hours, more than 2 hours, more than 3 hours, more than 4 hours or more than 5 hours, more than 6 hours, more than 7hours, more than 8 hours, more than 9 hours, more than 10 hours.
  • the rhBMP-9 of the present invention is preferably produced by a recombinant eukaryotic cell that is capable of being maintained under cell culture conditions.
  • a recombinant eukaryotic cell that is capable of being maintained under cell culture conditions.
  • Non-limiting examples of this type of cell are non-primate eukaryotic cells such as Chinese hamster ovary (CHOs) cells including CHO-derived Ser320-Arg429, CHO- K1 (ATCC CCL 61 ) cells and SURE CHO-M cells (derivative of CHO-K1 ), and baby hamster kidney cells (BHK, ATCC CCL 10).
  • CHOs Chinese hamster ovary
  • eukaryotic host cells include, e.g., human cervical carcinoma cells (HELA, ATCC CCL 2) and 293 [ATCC CRL 1573] as well as 3T3 [ATCC CCL 163] and monkey kidney CV1 line [ATCC CCL 70], also transformed with SV40 (COS-7, ATCC CRL-1587).
  • HELA human cervical carcinoma cells
  • 293 ATCC CRL 1573
  • 3T3 ATCC CCL 163
  • monkey kidney CV1 line ATCC CCL 70
  • COS-7 ATCC CRL-1587
  • RhBMPs can be administered in glycosylated (gBMP-9) or non-glycosylated form (ng BMP-9).
  • One or more glycosylations may be conferred to the rhBMP-9 protein when expressed in said eukaryotic cells. All examples set forth herein were performed with CHO-cell expressed rhBMP-9 as the rhBMP-9.
  • the glycosylation may be one or more glycosylation of an amino acid of the core domain.
  • the recombinant human rhBMPs comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, or more of the glycosylations of the native, endogenously-expressed BMP-9 in vivo from a human cell.
  • gBMP-9 is administered to a patient and/or is part of the composition/kit of the present invention.
  • the composition may or may not contain Bovine Serum Albumin (BSA), but in certain embodiments does not contain BSA which is used as a carrier protein to enhance protein stability, increases shelf-life, and/or allows the recombinant protein to be stored at a more dilute concentration.
  • BSA Bovine Serum Albumin
  • the rhBMP-9 may also comprise other post-translational modifications which may or may not result from expression in a eukaryotic cell, such as for example,
  • compositions/kits of the present invention or according to the method set forth herein include metabolic bone disease, osteoarthritis, osteochondral disease, rheumatoid arthritis, osteoporosis, bone fractures, Paget's disease, periodontitis, or
  • dentinogenesis can effectively treat a non-mineralized skeletal tissue disorder, injury or diseases selected from the group consisting of osteoarthritis, osteochondral disease or defect, chondral disease or defect, rheumatoid arthritis, trauma-induced and inflammation-induced cartilage
  • intervertebral discs degenerative diseases of the intervertebral disc (for example, degenerative disc disease), osteochondrosis, and injuries and diseases of ligament, tendon, synovial capsule, synovial membrane and meniscal tissues.
  • Certain other preferred embodiments can effectively treat tissue injury selected from the group consisting of: trauma-induced and inflammation-induced cartilage degeneration, articular cartilage injuries, full thickness cartilage defects, superficial cartilage defects, hierniation and rupture of intervertebral discs, degeneration of intervertebral discs due to an injury(s), and injuries of ligament, tendon, synovial capsule, synovial membrane and meniscal tissues.
  • compositions described herein or the kits comprising the constituents of the composition may comprise pharmaceutically acceptable excipient (s).
  • the compositions of the present invention are injected or implanted at a site of bone or tissue injury, disorder, or disease.
  • the composition is a transdermal composition.
  • the method of the present invention comprises administering to a subject/patient at a therapeutically appropriate site a pharmaceutically effective amount of the compositions described herein.
  • compositions described herein may also comprise a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant.
  • Acceptable formulation materials are, for example, nontoxic to recipients at the dosages and concentrations employed.
  • compositions may contain materials for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition.
  • Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCI, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic
  • the compositions/kits of the invention can be used effectively to treat skeletal diseases or injuries.
  • the preparations can be used to treat a bone fracture, such as an open fracture or a closed fracture.
  • the preparation is preferably injected at the fracture site.
  • the preparations can be administered by surgical implantation at the fracture site.
  • the composition contains the rhBMP-9 in combination with a suitable matrix or support as outlined above.
  • the rhBMP-9 composition of the invention can be used to treat a disease or injury resulting in cartilage degradation or a cartilage defect.
  • the composition can be applied to a cartilage defect site, such as a degenerative intervertebral disc, or other fibrocartilaginous tissue, including a tendon, a ligament or a meniscus.
  • a cartilage defect site such as a degenerative intervertebral disc, or other fibrocartilaginous tissue, including a tendon, a ligament or a meniscus.
  • composition of the invention can also be used to treat a defect or degeneration of articular cartilage, as set forth in published PCT application WO 05/1 15438 which is incorporated herein by reference in its entirety, such as the cartilage lining of a joint, such as a synovial joint, including a knee, an elbow, a hip, or a shoulder.
  • the composition is preferably injected into the synovial space of the joint.
  • compositions of the invention are used to treat an articular cartilage defect site, such as a chondral defect or an osteochondral defect.
  • articular cartilage defects can be the result of a disease process, such as osteoarthritis or rheumatoid arthritis, or due to injury of the joint.
  • the composition can be injected into the joint space or it can be surgically implanted.
  • the rhBMP-9 can be placed within the defect either alone or in combination with one or more additional active agents, a supporting matrix or support and/or marrow stromal cells.
  • compositions of the present invention can be administered in several different ways (see, e.g., US Patent Publ. 20140200182, which is incorporated herein by reference in its entirety).
  • the patient has a bone defect requiring a bone graft.
  • the bone defect cavity is filled with a fibrin sealant such as TISSEEL, comprising less than 12mg of rhBMP-9.
  • An autologous bone graft having a total volume of less than one third of the total bone defect is implanted by a surgeon into the bone defect cavity.
  • Rh-BMP-9 stimulates bone formation so that the bone regenerates in the bone defect cavity. After 6 to 9 months the autologous bone graft is surrounded by regenerated bone of the patient.
  • a patient in need of a dental implant and who has experienced bone loss due to, e.g., gum disease or trauma and thus does not have enough bone structure to support and stabilize an implant is treated.
  • a kit comprising a resorbable collagen sponge and rhBMP-9 is provided and the collagen sponge is coated with rhBMP-9, implanted at the point of bone loss (as in a sinus lift site, an extraction site, or on a deficient dental ridge) and new bone formation occurs.
  • an implant such as an autogenous bone graft that fills less than 50% of the space exists due to the bone loss is inserted at the site.
  • the bone is allowed to grow for several months.
  • This autogenous bone graft fuses with the existing bone and the migration of cells causes firm adhesion and cell growth.
  • the rhBMP-9 is also resorbed after it completed the task of initiating the normal bone healing process.
  • sequence identity refers to a measure of the identity of nucleotide sequences or amino acid sequences. In general, the sequences are aligned so that the highest order match is obtained. "Identity", per se, has recognized meaning in the art and can be calculated using published techniques. (See, e.g.: Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988;
  • amino acid sequence is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance SEQ ID NO: 1 , or a part thereof (see, in particular parts identified above), can be determined conventionally using known computer programs such the BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University
  • BESTFIT uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981 ), to find the best segment of homology between two sequences.
  • the parameters are set such that the percentage of identity is calculated over the full length of the reference nucleic acid or amino acid sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.
  • sequence identity relates to the sum of all the residues specified.
  • rh-BMP-9 molecules that contain "conservative substitutions.”
  • residues that are physically or functionally similar to the corresponding reference residues are substituted for the same. That is, 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. (1978), 5 Atlas of Protein Sequence and Structure, Suppl. 3, Ch. 22, pp. 354-352, Natl. Biomed. Res. Found., Washington, D.C. 20007.
  • 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, methionine; and (h) phenylalanine, tyrosine.
  • rhBMP-2 and -9 were examined at the various indicated concentrations with membranes for cell attachment at 8 hours post-seeding. It was found that none of the investigated concentrations of either rhBMP-2 or rhBMP-9 had any significant influence on cell attachment. Thereafter, and as shown in Fig. 3, cell proliferation was determined at 1 , 3 and 5 days post seeding. No significant differences between the 5 groups were observed for cell
  • Figs. 4 and 5 the effects of both concentrations of rhBMP-2 and -9 indicated were then examined on osteoblast differentiation by real-time PCR and alizarin red staining. All concentrations of either rhBMP-2 or rhBMP-9 had no effect on mPiNA levels of Runx2 at either 3 or 14 days post seeding when compared to control samples (Fig. 4A). However, all concentrations of rhBMPs significantly upregulated ALP expression at 3 days and BSP expression at 14 days when compared to control samples (Fig. 4B, C).
  • rhBMP9 high group demonstrated significantly higher ALP mRNA levels approximately 5 fold when compared to all other groups at 3 days and also significantly induced ALP levels 2 fold at 14 days when compared to control group (Fig. 4B).
  • alizarin red staining revealed higher staining intensity for rhBMP-9 samples when compared to their respective rhBMP-2 groups (rhBMP-9 high group versus rhBMP-2 high group; rhBMP-9 low group versus rhBMP-2 low group, Fig. 5).
  • collagen membranes provide 3 dimensional frameworks to stimulate osteoblast behavior. It was found that cell attachment to collagen membranes was similar on all tested samples including those pre-coated with either rhBMP-2 or rhBMP-9 at both concentrations tested with little differences observed on cell proliferation (Fig. 2). In general, it was thus observed that collagen membranes utilized alone were able to facilitate cell attachment and promote their proliferation up to a 5-day period (Fig. 3).
  • rhBMP-2 and rhBMP-9 had the greatest influence on osteoblast differentiation.
  • rhBMP-2 in combination with a collagen membrane was able to significantly upregulate the differentiation of osteoblasts in vitro ((Miron et al., 2013).
  • rhBMP-9 remarkably promoted osteoblast differentiation on membranes when compared to rhBMP-2 (Fig. 4, 5).
  • Both rhBMP-2 and rhBMP-9 induced significantly higher mRNA levels of ALP at 3 days and BSP levels at 14 days when compared to control samples, while only high concentration of rhBMP-9 significantly increased ALP expressions compared to any other modalities at 3 and 14 days (Fig. 4).
  • rhBMP-9 demonstrated significantly higher staining of alizarin red when compared to rhBMP-2, yet did not demonstrate any differences in BSP mRNA levels, a late differentiation marker for osteoblasts. Many different hypotheses for this finding have been considered. It is clear that the effects of either rhBMP took action at earlier time points as visualized by the ALP mRNA expression at 3 days when compared to 14 days (Fig. 4B).
  • Recombinant human rhBMP-2 Recombinant Human/Mouse/Rat BMP-2 Protein, CHO-derived Gln283-Arg396, Accession no. P12643, Cat. No. 355-BM as of Jan. 2016
  • rhBMP-9 CHO-derived Ser320-Arg429, Accession no. Q9UK05, Cat. No. 3209-BP as of Jan. 2016
  • porcine collagen membrane (BIOGIDE, GEISTLICH PHARMA, CH) alone, 2) rhBMP-2 low (10 ng/ml) + membrane, 3) rhBMP-2 high (100 ng/ml) + membrane, 4) rhBMP-9 low (10 ng/ml) + membrane, and 5) rhBMP-9 high (100 ng/ml) + membrane were examined. Scanning electron microscopy (SEM) was utilized to visual the 3- dimentional topography of the membranes utilized in the present study (Fig. 1 ).
  • Undifferentiated mouse cell-line ST2 (RIKEN cell bank, JP) were cultured in a humidified atmosphere at 37°C in growth medium consisting of DMEM
  • NBM natural bone mineral
  • BIO-OSS natural bone mineral
  • rhBMP9 concentration (100 ng/ml) of rhBMP9 with NBM were examined.
  • Undifferentiated mouse cell-line ST2 was obtained from RIKEN Cell Bank (Tsukuba, Japan) and therefore no ethical approval was necessary for the present study.
  • Cells were cultured in a humidified atmosphere at 37°C in growth medium consisting of DMEM (Invitrogen Corp., Carlsbad, CA), 10% fetal Bovine serum (FBS; Invitrogen Corp.), and antibiotics (Invitrogen Corp.).
  • DMEM Invitrogen Corp., Carlsbad, CA
  • FBS fetal Bovine serum
  • antibiotics Invitrogen Corp.
  • ST2 cells were seeded in 24-well plates at a density of 10,000 cells per well either control NBM/membrane or with NBM/membrane + rhBMP-2 or -9.
  • Cells were quantified using fluorescent MTS assay (PROMEGA, Madison, Wl) using an ELx808 Absorbance Reader (BIO-TEK, Winooski, VT) at 1 , 3 and 5 days for cell proliferation as previously described (Miron et al., 2013b).
  • PBS phosphate buffered solution
  • BIO-TEK fluorescence plate reader
  • Primer and probe sequences for genes encoding runt-related transcription factor 2 (Runx2), collagen 1 a2 (COL1 A2), alkaline phosphatase (ALP), osteocalcin (OC) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were fabricated with Primer sequences according to Table 1 .
  • Real-time RT-PCR was performed using Roche Master mix and quantified on an Applied Biosystems 7500 Real-Time PCR Machine.
  • a Nanodrop 2000c (Thermo, Wilmington, DE) was used to quantify total RNA levels.
  • the AAC ⁇ method was used to calculate gene expression levels normalized to total RNA values and calibrated to control samples.
  • ST2 cells were seeded in 24-well plates at a density of 50,000 cells per well with either control substrate/matrix or with substrate/matrix+ rhBMP-2 or -9 in osteogenic differentiation medium (ODM), which consisted of DMEM supplemented with 10% FBS, 1 % antibiotics, 50 ⁇ g/ml ascorbic acid (Sigma, St. Louis, MO) and 10 mM ⁇ - glycerophosphate (Sigma) to promote osteoblast differentiation as previously described (Miron et al., 201 1 ). Alizarin red staining was performed to determine the presence of extracellular matrix mineralization. After 14 days, cells were fixed in 96% ethanol for 15 minutes and stained with 0.2% alizarin red solution (SIGMA, St.
  • ALP alkaline phosphatase
  • alkaline phosphatase activity was monitored using Leukocyte alkaline phosphatase kit (procedure No. 86, Sigma). ST2 cells were fixed by immersing in a citrate-acetone-formaldehyde fixative solution for 5 min and rinsed in deionized water for 1 min.
  • Alkaline dye mixture are prepared by 1 ml Sodium Nitrite Solution and 1 ml fast red violet alkaline solution dissolved in 45 ml of distilled water and 1 ml of Naphtol AS-BI alkaline solution. Surfaces were then placed in alkaline dye mixture solution for 15 min protected from light.
  • Rabbits were anesthetized intramuscularly with Premedication included Ketamin 65 mg/kg (Vetoquinol AG, Bern, Switzerland) and Xylazin 4 mg/kg s.c. (Vetoquinol AG) in neck wrinkles (pain free). Narcosis was maintained with Ketamin 130 mg/kg (Vetoquinol AG) and Xylazin 8 mg/kg in 100 mL NaCI i.v. (Vetoquinol AG) under spontaneous breading of O2 by the mask. Intraoperative analgesia was achieved with Fentanyl plaster 2.1 mg (Janssen Cilag AG, Baar, Switzerland).
  • Surgical area was desinfected and a straight incision was made from the nasal bone to the midsagittal crest. The soft tissues were reflected and the periosteum was elevated from the site.
  • four evenly distributed 6mm diameter craniotomy defects were prepared with a trephine bur under copious irrigation with sterile saline. Care was exercised to avoid injury of the dura. The surgical area was flushed with saline to remove bone debris.
  • the following 6 treatment modalities were randomly allocated to all 48 defects: (i) NC (negative control, empty), (ii) BO (Bio-Oss®, S-size; Geistlich Pharma AG, Wolhusen,
  • defect sites were subjected to radiography (25 kVP for 10 sec) in two projections using a desktop Cone-Beam scanner (microCT 40, Scanco Medical AG,
  • the X-ray source was set at 70 kVp with 1 14 mA at high resolution (1000 projections/1808), which showed an image matrix of 2048 3 2048 pixels. Integration time was set on 3s. Micro-CT images were then reconstructed using 3D structural analysis software (Amira, Visualization Sciences Group,
  • the region of interest was selected corresponding to the dimensions of the defect sites, with a diameter of 6 mm full-thickness cylinders and then new bone formation (BV, mm3) was measured.
  • TISSEELS DUO QUICK from BAXTER A/S (Aller, Denmark), comprises two 1 -mL syringes.
  • One syringe contains fibrinogen, fibronectin, bovine aprotonin, factor XIII, and plasminogen.
  • the other syringe contains human thrombin (500 IU) and 40 mmol CaC .
  • Tisseels was prepared according to the manufacturer's instructions. The protein concentration of Tisseels is 50-65 mg/mL according to the manufacturer's product specification. Tisseel is utilized to stabilize bone grafting materials due to the fact the fibrin solidifies within seconds following its application.
  • Hyaluronic acid gel carrier system by REGEDENT is a Hyaluronic acid gel carrier system by REGEDENT
  • hyaDENT BG a sterile gel based on hyalorunic acid of non- animal source was obtained. Its use is commonly applied for soft and hard tissue regeneration in the dental field.
  • the material composition includes per ml: Sodium hyaluronate 2.0 mg, sodium hyaluronate crosslinked 16.0 mg, sodium
  • Collacone® is a wet-stable and moldable cone made of natural collagen. As a completely resorbable and hemostatic wound coverage, it is intended for application in fresh extraction sockets in the daily clinical practice.
  • a bovine derived mineral that includes collagen (Hypro-Oss®, Biolmplon):
  • Hypro-Oss® is a lyophilized natural bovine composite of hydroxyapatite and atelocollagen type I, which is indicated for support new bone formation and reconstruction of bone defects.
  • Native composite approx. 70% hydroxyapatite Ca5(P04)3(OH) and 30% atelocollagen type I bovine origin in the form of bone granules of two different grain sizes: 0.5 - 1 mm and 1 -2 mm.
  • a demineralized freeze-dried allograft from human origin (DBX):
  • DBX is utilized as one of the only available materials in Europe coming from human origin. DBX is sterilized under strict guidelines approved for use by the FDA and CE.
  • a synthetic bone graft fabricated from hydroxyapatite (MAXRESORB, Botiss):
  • MAXRESORB® is an innovative, safe, reliable, and fully synthetic bone substitute material that is characterized by controlled resorption properties and outstanding handling characteristics composed of 60% hydroxyapatite (HA) and 40% beta- tricalcium phosphate ( ⁇ -TCP).
  • HA 60% hydroxyapatite
  • ⁇ -TCP beta- tricalcium phosphate
  • the unique synthesis-based production process ensures a completely homogenous distribution of both mineral phases.
  • the peculiar composition of MAXRESORB® promotes the fast formation of new vital bone, ensuring a long-term mechanical and volume stability.
  • the osteoconductivity of MAXRESORB is achieved by a matrix of interconnecting pores (with a size ranging between 200 and 800 ⁇ ) and a very high porosity of approx. 80%.
  • the high microporosity and nano-structured surface facilitate the uptake and adsorption of blood, proteins, and stem cells.
  • the macropores are ideal for the ingrowth of osteogenic cells and the bony integration.
  • MAXRESORB was also utilized as a bone block in 1 in vitro study.
  • MAXRESORB Fabricated from the same bone grafting components as MAXRESORB (above) however includes a paste that is highly viscous and allows the perfect shaping, molding, fitting, and complete bone bonding to the surrounding bone surface of the defect.
  • MAXRESORB® inject is a non-hardening synthetic bone paste.
  • JASON membrane is a native collagen membrane obtained from porcine
  • pericardium developed and manufactured for dental tissue regeneration.
  • the superior biomechanical and biologic properties of the natural pericardium are preserved during the patented production process.
  • the JASON membrane exhibits excellent handling characteristics like a remarkable tear resistance and very good surface adaptation. Due to the natural comb-like and multilayered collagen structure with an increased content of collagen type II I shows a slowed degradation. Therefore, JASON membrane offers a prolonged barrier function and is the first choice particularly for larger augmentative procedures.
  • MUCODERM is a natural type l/lll collagen matrix derived from porcine dermis that undergoes a multi-stage purification process, which removes all potential immunogens. The remaining matrix is a membrane that consists of collagen and elastin. MUCODERM promotes the revascularization and fast soft tissue integration and is a valid alternative to the patient's own connective tissues. After placement, the patient's blood infiltrates the MUCODERM graft through the three-dimensional network, bringing host cells to graft surface and triggering the revascularization process. A significant revascularization may begin after the implantation depending on the health condition of the patient.
  • Kang et al. Characterization of the distinct orthotopic bone-forming activity of 14 BMPs using recombinant adenovirus-mediated gene delivery; Gene Therapy 2004 July 22, 1 1 , 1312-1320.
  • Non-glycosylated BMP-2 can induce ectopic bone formation at lower concentrations compared to glycosylated BMP-2, J Control Release 2012 Apr 10; 159(1 ): 69-77.
  • Gin Lys His lie Leu Leu Phe Asn lie Ser lie Pro Arg His Glu Gin 130 135 140 lie Thr Arg Ala Glu Leu Arg Leu Tyr Val Ser Cys Gin Asn His Val 145 150 155 160
  • Arg Glu Met lie Ser His Glu Gin Glu Ser Val Leu Lys Lys Leu Ser
  • Thr Pro Thr Lys His Ala lie Val Gin Thr Leu Val His Leu Lys Phe 370 375 380

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Abstract

L'invention concerne des compositions, des nécessaires et des méthodes pour favoriser la croissance et/ou la régénération osseuse chez des patients en ayant besoin par l'intermédiaire de rhBMP-9.
PCT/IB2016/053147 2016-05-27 2016-05-27 Composition et méthodes pour l'ingénierie des tissus osseux WO2017203331A1 (fr)

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US11696974B2 (en) 2019-05-30 2023-07-11 Sichuan University Method for preparing a functionally gradient material for guided periodontal hard and soft tissue regeneration

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