WO2017203331A1

WO2017203331A1 - Composition and methods for bone tissue engineering

Info

Publication number: WO2017203331A1
Application number: PCT/IB2016/053147
Authority: WO
Inventors: Richard MIRON; Matthias MOTTINI
Original assignee: Universität Bern
Priority date: 2016-05-27
Filing date: 2016-05-27
Publication date: 2017-11-30

Abstract

Disclosed are compositions, kits and methods for promoting bone growth and/or regeneration in patients in need thereof via rhBMP-9.

Description

COMPOSITION AND METHODS FOR BONE TISSUE ENGINEERING

FIELD OF THE INVENTION

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.

BACKGROUND

Bone formation is a well-orchestrated process of osteoblast lineage-specific differentiation. During osteogenesis, 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. Among graft materials, autologous bone grafts having osteoconductivity, osteoinductivity, and osteogenecity are widely used for the treatment of bone repair and regeneration.

Despite many advantages of autologous bone grafts, shortcomings associated with their usage include, among others, limited supply, donor-site morbidity and additional surgical times, which restrict their use in clinical practice. Thus, in clinical practice autologous bone grafts are often replaced with artificial bone graft materials including metals, ceramics such as hydroxyapatite (HAp), β-tricalcium phosphate (β-TCP), and biphasic calcium phosphate (BCP), or collagen materials.

For effective bone repair and regeneration, osteogenesis needs to be induced after the graft material has been implanted. Thus, the graft material must not only have a three-dimensional, often porous structure, but also be able to mimicry the

extracellular matrix (ECM) in order to produce bone tissue. To this end the graft material is combined with growth factors, in particular BMPs. The administration of recombinant BMPs, including human recombinant (rh) BMPs, often fails in view of short biological half-lives, the lack of matrixes which allow for controlled and sustained BMP delivery at the site of implantation, and the inability of recombinant molecule presentation after implantation to mimic the route of administration in vivo by a BMP-producing cell. In view of this, authors have in the past suggested gene-therapy based approaches to optimally deliver BMPs.

{Franceschi et al. 2000). However, such approaches have not received regulatory approval. In 2002, the U.S. Food and Drug Administration (FDA) approved for the first time a BMP for clinical use. Two BMP products are commercially available since 2003, rhBMP-2 (INFUSE, MEDTRONIC, Memphis, Tenn.) and BMP-7 (OP-1 PUTTY, STRYKER, Kalamazoo, Mich.). 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

compromised patients. Despite this regulatory milestone for in particular BMP-2, its use is often not practical due to a high dosage requirements and associated costs {Boden et al. 2000). A 15,000-fold higher concentration of rhBMP-2 is required to induce bone growth in humans (1 .5 mg/ml, e.g., a total dose of 12 mg) than in cell culture (100 ng/ml) (Govender et al., 2002). Despite the excellent spinal fusion rates obtained with rhBMP-2, a multitude of secondary side effects have been associated with its use including retrograde ejaculation, antibody formation, postoperative radiculitis, postoperative nerve root injury, ectopic bone formation, vertebral osteolysis/edema, dysphagia and neck swelling, hematoma formation, interbody graft lucency, and wound healing complications {Tannoury et. al. 2014).

To overcome the limitations of 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).

Another approach was the use of non-glycosylated (ng) 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. NgBMP-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. Upon ectopic implantation, 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).

At least 15 types of BMPs have been identified in humans. Analysis of the osteogenic activity of 14 types of human BMPs (i.e. BMP-2 to BMP-15) using recombinant adenovirus-mediated gene delivery, found that BMP-2, BMP-6, and BMP-9 were the most potent factors, promoting osteogenic differentiation of mesenchymal stem cells both in vitro and in vivo. Kang et al. reported that their recombinant adenoviral (ad) vectors transduced osteoblast progenitor cells with high efficiency to continuously produce biologically active BMPs inside the mammalian cells. Under these advantageous conditions, 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. Thus, 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. Within the mature protein, 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 . In the embryonal CNS, 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

concentrations, it enhances TGF-beta 1 production and inhibits hematopoietic progenitor colony formation.

There remains a need for a BMP, preferably a rhBMP, that can be administered at doses lower than rh-BMP-2 to limit secondary side effects. There is further a need for 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. There is further a need for compositions, in particular pharmaceutical compositions, containing BMPs, in particular rhBMPs that allow controlled and sustained BMP delivery at a site of implantation.

The present invention addresses one or more of those needs and/or other needs in the art.

BRIEF SUMMARY OF THE INVENTION

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:

(a) optionally, at least one substrate and/or matrix, and

(b) 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 present invention is also directed to a composition or kit for promoting bone growth and/or inhibiting bone loss.

The composition or kit may comprise

(a) rhBMP-9, wherein the 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

wherein the 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

(b) an amino acid sequence comprising:

- amino acids 318 to 429 or 320 to 429 of SEQ ID NO: 1 or a sequence having 80%, 90%, 95, 98, 99% or complete sequence identify with amino acids 318 to 429 of SEQ ID NO: 1 , or

at least 80, 90, 100 consecutive amino acids of amino acids 318 to 429 of SEQ ID NO: 1 ; and, optionally, one or more three- dimensional substrates or matrixes. The rhBMP-9 of (a) or the amino acid sequence of (b) may comprise one or more posttranslational modifications, such as glycosylations, which may have been conferred by expression in an eukaryotic cell. A kit 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. 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 (ε-caprolactone) (PCL), or co-polymers and/or combinations thereof. The 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.

BRIEF DESCRIPTON OF THE FIGURES

Figure 1 shows SEM (scanning electron microscope) images of collagen

membranes at (left) low and (right) high magnification. Noticeable are the collagen fibrils on the surface of the porcine derived membranes.

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). 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). In equivalent experiments comprising NBM bone grafts seeded on 1 ) 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 (100ng/ml), significant differences were observed between rhBMP-9 (high) and (i) rhBMP-2 (low) and the control.

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. (B) Proliferation assay of ST2 cells seeded on (i) control (Plastic dish), (ii) TISSEEL only, (iii) TISSEEL + rhBMP-9 (100 ng/ml) at 1 , 3 and 5 days post-seeding. It was found that TISSEEL + rhBMP-9 demonstrated significantly lower cell numbers at 3 days post-seeding when compared to on plastic dish samples. No significant differences were observed between TISSEEL only and TISSEEL + rhBMP-9 at 1 , 3 and 5 days post-seeding. (^*denotes significant difference, P < 0.05). The assays were run in triplicate.

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. (B) Quantified data of alizarin red staining from color thresholding software for ST2 cells (^** denotes significantly higher than all other treatment modalities, p<0.05). RhBMP-9 significantly increased Alizarin red staining when compared with control plastic and TISSEEL only samples. The assays were performed in triplicate.

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. Either HA only and HA + rhBMP-9 samples demonstrated significantly lower cell proliferation when compared to on plastic dishes while no significant differences were observed between the 2 HA groups at 1 , 3 and 5 days post cell seeding, ("denotes significantly higher than all other modalities, P < 0.05). The assays were run in triplicate.

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. (B) Quantified data of alizarin red staining from color thresholding software (^* denotes significant difference, p<0.05, ^** denotes significantly higher than all other treatment modalities, p<0.05). 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. (B) Proliferation assay of ST2 cells seeded on (i) 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 1 , 3 and 5 days post- seeding. No significant differences were observed between 6 groups, ("denotes significantly higher than all other modalities, P < 0.05). The assays were run in triplicate.

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. (B) 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-2 high and rhBMP-9 low samples significantly induced higher ALP staining when compared with collacone only samples. rhBMP-9 high samples significantly promoted ALP staining when compared with all other modalities. The assays were performed in triplicate.

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. (B) Quantified data of alizarin red staining from colour thresholding software for ST2 cells (^* denotes significant difference, p<0.05, ^** denotes significantly higher than all other treatment modalities, p<0.05). Collacone + rhBMP-2 high and rhBMP-9 low groups demonstrated significantly higher Alizarin red staining when compared to control groups. rhBMP-9 high group significantly increased Alizarin red staining when compared with all other modalities. The assays were performed in triplicate.

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. With rhBMP-2 high, rhBMP-9 low, or rhBMP high groups significantly lower cell adhesion on Bio-Oss® were observed relative to the control plastic samples, whereas no significant differences were observed between Hypro- Oss® groups at 8 hrs post cell seeding. (B) Proliferation assay of ST2 cells seeded on (i) a control (Plastic dishes), (ii) Bio-Oss® only, (iii) Bio-Oss® + rhBMP-2 low (10 ng/ml) (iv) Bio-Oss® + rhBMP-2 high (100 ng/ml), (v) rhBio-Oss + rhBMP-9 low (10 ng/ml) (vi) Bio-Oss® + rhBMP-9 high (100 ng/ml) at 1 , 3 and 5 days post-seeding. Significantly higher cell proliferation was observed on control plastic dish samples when compared with any other Bio-Oss® groups at 1 , 3 and 5 days post cell seeding, while no significant differences were observed between all 5 Bio-Oss® groups. (C) Proliferation assay of ST2 cells seeded on (i) a control (Plastic dishes), (ii) Hypro-Oss® only, (iii) Hypro-Oss® + rhBMP-2 low (10 ng/ml) (iv) Hypro-Oss® + rhBMP-2 high (100 ng/ml), (v) Hypro-Oss® + rhBMP-9 low (10 ng/ml) (vi) Hypro- Oss® + rhBMP-9 high (100 ng/ml) at 1 , 3 and 5 days post-seeding. No significant differences were observed between all 5 Hypro-Oss® groups at 1 , 3 and 5 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 22 shows the results of alkaline phosphatase (ALP) staining of ST2 cells treated at 7 days post-seeding. (A) 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). Weblike structures formed on Bio-Oss particles previously coated with rhBMP-9 either at low or high dose. (B) ALP staining of Hypro-Oss particles at 7 days post-seeding with (i) Hypro-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). Higher ALP staining was found on Hypro-Oss® particles previously coated with rhBMP-9 either at low or high dose. (C) 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). 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. (C) Quantified data of alizarin red staining from color thresholding software (^* denotes significant difference, p<0.05, ^** denotes significantly higher than all other treatment modalities, p<0.05). rhBMP-9 coated bone graft particles significantly increased Alizarin red staining either on Bio- Oss® or Hypro-Oss®. The assays were performed in triplicate. 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

demonstrated significantly lower cell adhesion either on DBX or MAXRESORB when compared with control plastic dishes at 8 hrs post cell seeding. (B) Proliferation assay of ST2 cells seeded on (i) Control (Plastic dishes), (ii) DBX only, (iii) DBX + rhBMP-2 low (10 ng/ml) (iv) DBX + rhBMP-2 high (100 ng/ml), (v) DBX + rhBMP-9 low (10 ng/ml) (vi) DBX + BMP-9 high (100 ng/ml) at 1 , 3 and 5 days post-seeding. Significantly higher cell proliferation was observed on control plastic dishes when compared with all other groups at 1 , 3 and 5 days post cell seeding, whereas no significant differences were observed between all 5 DBX groups. (C) Proliferation assay of ST2 cells seeded on (i) Control (Plastic dishes), (ii) MAXRESORB only, (iii) MAXRESORB + rhBMP-2 low (10 ng/ml) (iv) MAXRESORB + rhBMP-2 high (100 ng/ml), (v) MAXRESORB + rhBMP-9 low (10 ng/ml) (vi) MAXRESORB + rhBMP-9 high (100 ng/ml) at 1 , 3 and 5 days post-seeding. Significantly higher cell

proliferation was observed on control plastic dishes when compared with all other groups at 1 and 5 days post cell seeding, while no significant differences were observed between all 5 MAXRESORB groups at 1 , 3 and 5 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 25 shows the results of alkaline phosphatase (ALP) staining of ST2 cells treated at 7 days post-seeding. (A) 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),

(v) rhBMP-9 low (10 ng/ml) (vi) rhBMP-9 high (100 ng/ml). (B) ALP staining of MAXRESORB particles at 7 days post-seeding with (i) MAXRESORB 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). 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, whereas 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. (B) Proliferation assay of ST2 cells seeded on (i) Control (Plastic), (ii) Jason only, (iii) Jason + rhBMP-2 (100 ng/ml) and (iv) Jason + rhBMP-9 (100 ng/ml) at 1 , 3 and 5 days post-seeding. (C) Proliferation assay of ST2 cells seeded on (i) Control (Plastic), (ii) MUCODERM only, (iii) MUCODERM + rhBMP-2 (100 ng/ml) and (iv) MUCODERM + rhBMP-9 (100 ng/ml) at 1 , 3 and 5 days post- seeding. Either membrane group demonstrated significantly lower cell proliferation when compared to on plastic dishes, whereas no significant differences were observed between membrane samples at 1 , 3 and 5 days post-seeding, ("denotes significantly higher than all other modalities, P < 0.05). The assays were run in triplicate.

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

demonstrated significantly higher intensity of Alizarin red staining when compared with control JASON membrane only. (D) MUCODERM coated with rhBMP-2 and rhBMP-9 induced significantly higher Alizrin red staining when compared with MUCODERM only. The assays were performed in triplicate.

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_5μg, and (vi) BO + rhBMP-9_20μg were applied into bone defects and covered with BioGide®. (B) Typical micro-CT images of (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_5μg, and (vi) BO + rhBMP-9_20μg at 8 weeks post surgery. (C) New bone volume (BV) was measured at bone defect area. Bio-Oss® coated either with rhBMP-9_5μg or rhBMP-9_20μg 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).

DETAILED DESCRIPTION OF THE INVENTION

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. In many cases, 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. For example, a substrate is a matrix if the rhBMP-9 is directly applied onto the substrate. However, a matrix might be applied to a substrate and in turn carry the rhBMP-9. In a bone graft coated with collagen and rhBMP-9, the bone graft is the substrate and the collagen is the matrix. In a bone graft directly coated with rhBMP-9, 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.

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 (BioOss™) or a natural bovine derived bone mineral (e.g.,Hypro-Oss™). 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). In one preferred embodiment 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). 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. The

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 Ca²⁺ may catalyze polymerization of the fibrinogen, converting the fibrinogen into fibril polymer. Further, thrombin and Ca²⁺ 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

(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,

orthophosphates, beta-tricalcium phosphates, alpha-tricalcium phosphates or combinations thereof. The substrate or matrix may in addition or alternatively comprise polyesters such as poly(glycolic acid) (PGA), poly(lactic acid) (PLA), poly (ε-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. In the context of tissue engineering, 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.

As described further herein, 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

(demineralized bone matrix), 7) synthetic bone grafts, e.g., fabricated from hydroxyapatite such as MAXRESORB (Botiss), 8) injectable synthetic pastes containing bone particles such as MAXRESORB Inject (Botiss) and 9) different types of collagen barrier membranes.

As the person skilled in the art will appreciate, different approaches exist as to how rhBMP-9 may be loaded onto the matrix/substrate. In its simplest form, 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 . Materials that precipitate, such as hydroxyapatite (HA), also allow for incorporation during precipitation. Materials that undergo conversions, also allow for incorporation during this conversion (e.g., dicalcium phosphate dihydrate (DCPD) conversion to HA). 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

(Fluorotechnics, Sydney, Australia) and the Quantikine (R&D Systems Inc,

Minneapolis, Minn) rhBMP-9 enzyme-linked immunosorbent assay (ELISA) can be used to determine total protein uptake into the matrix/substrate and its release profile in vitro. Preferably, 50-100%, preferably more than 60%, 70%, 80% or 90% of the rhBMP-9 provided are incorporated.

Preferably 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. Depending on the incorporation method there might be one, two or more burst releases during that time, e.g., after 12 hours and, e.g., 7 days.

In the matrix/substrate/ rhBMP-9 compositions of the present invention, 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"). Generally, 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. In certain embodiments of the invention the parameters for osteoblast differentiation, in particular ALP staining showed absolute values over 150%, over 200%, over 300%. Interestingly, 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

concentrations of 100ng/ml was loaded simultaneously in a 3rd syringe so that the entire matrix would harden with the rhBMP-9 contained within its scaffold, e used 100ng/ml. The Figures show that TISSEEL in combination with rhBMP-9

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.

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. Furthermore, 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. Thus, the mature peptide associated with the propeptide may also be administered in accordance to the present invention.

SEQ ID NO: 1 :

MCPGALWVAL PLLSLLAGSL QGKPLQSWGR GSAGGNAHSP LGVPGGGLPE (50)

HTFN LKMFLE NVKVDFLRSL N LSGVPSQDK TRVEPPQYM I DLYNRYTSDK (100)

STTPASNIVR SFSM EDAISI TATEDFPFQK HI LLFN ISI P RH EQITRAEL (150)

RLYVSCQNHV DPSHDLKGSV VIYDVLDGTD AWDSATETKT FLVSQDIQDE (200)

GWETLEVSSA VKRWVRSDST KSKNKLEVTV ESHRKGCDTL DISVPPGSRN (250)

LPFFVVFSN D HSSGTKETRL ELREM ISHEQ ESVLKKLSKD GSTEAG ESSH (300)

EEDTDGHVAA GSTLARRKRS AGAGSHCQKT SLRVNFEDIG WDSWIIAPKE (350)

YEAYECKGGC FFPLADDVTP TKHAIVQTLV HLKFPTKVGK ACCVPTKLSP (400)

ISVLYKDDMG VPTLKYHYEG MSVAECGCR (429) According 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. In one embodiment, a therapeutically effective dose of rhBMP-9 is less than the currently acceptable therapeutically effective amount for rhBMP-2. Currently, 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. In certain embodiments, 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. 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). Primate 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). The term recombinant signifies a cell that has been altered, e.g., by transfection with, e.g., a transgenic sequence encoding SEQ ID NO: 1 or parts thereof.

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. In one aspect of the present invention 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. In certain embodiments at least one of such glycosylation is missing. The term gBMP-9 includes partially and fully (= wild type) glycosylated forms of rh-BMP-9. In a preferred embodiment of the present invention, 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.

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,

phosphorylation, PEGylation, farnesylation, acetylation, biotinylation, lipidation (amino acid conjugated with lipid), and/or conjugation with an organic derivatizing agent.

Skeletal tissue disorders, injury or disease that may be treated with the

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. Other preferred embodiments 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

degeneration, age-related cartilage degeneration, articular cartilage injuries and diseases, full thickness cartilage defects, superficial cartilage defects, sequelae of systemic lupus erythematosis, sequelae of scleroderma, periodontal tissue regeneration, hierniation and rupture of 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.

The compositions described herein or the kits comprising the constituents of the composition may comprise pharmaceutically acceptable excipient (s). In some embodiments, the compositions of the present invention are injected or implanted at a site of bone or tissue injury, disorder, or disease. In some embodiments, the composition is a transdermal composition.

In some embodiments, 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 (or its constituents in a kit) 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. The resulting

pharmaceutical formulations 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 polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, polyethylene glycol (PEG), sorbitan esters, polysorbates such as polysorbate 20 and polysorbate 80, Triton, trimethamine, lecithin, cholesterol, or tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, e.g., sodium or potassium chloride, mannitol, or sorbitol);

delivery vehicles; diluents; excipients and/or pharmaceutical adjuvants. See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, 18.sup.th Edition, (A. R. Gennaro, ed.), 1990, Mack Publishing.

As noted above, the compositions/kits of the invention can be used effectively to treat skeletal diseases or injuries. For example, the preparations can be used to treat a bone fracture, such as an open fracture or a closed fracture. For the treatment of a closed fracture, the preparation is preferably injected at the fracture site. For open fractures, critical size defects or persistent non-unions, the preparations can be administered by surgical implantation at the fracture site. In most cases the composition contains the rhBMP-9 in combination with a suitable matrix or support as outlined above.

In one embodiment, the rhBMP-9 composition of the invention can be used to treat a disease or injury resulting in cartilage degradation or a cartilage defect. For example, 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. Such methods are set out in U.S. Pat. No. 6,958, 149 which is incorporated herein by reference in its entirety.

The 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. In this embodiment, the composition is preferably injected into the synovial space of the joint.

In another embodiment, the compositions of the invention are used to treat an articular cartilage defect site, such as a chondral defect or an osteochondral defect. Such articular cartilage defects can be the result of a disease process, such as osteoarthritis or rheumatoid arthritis, or due to injury of the joint. In this embodiment, the composition can be injected into the joint space or it can be surgically implanted. For example, 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.

The 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).

In another embodiment of the invention, 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.

In yet another embodiment, 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. Then 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.

The term 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;

Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991 ). While there exist a number of methods to measure identity between two

polynucleotide or polypeptide sequences, the term "identity" is well known to skilled artisans (Carillo, H. & Lipton, D., SIAM J Applied Math 48:1073 (1988)).

Whether the 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

Research Park, 575 Science Drive, Madison, Wis. 5371 1 ). 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.

When using DNAsis, ESEE, BESTFIT or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present invention, 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.

If, in the context of the present invention, reference is made to a certain sequence identity with a combination of residues of a particular sequence, this sequence identity relates to the sum of all the residues specified.

Included herein are rh-BMP-9 molecules that contain "conservative substitutions." Here, 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. 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, methionine; and (h) phenylalanine, tyrosine.

IN VITRO STUDIES

The in vitro studies outlined below made clear that even low concentrations of rhBMP-9 have similar or better osteopromotive potential relative to a 10 times higher dose of rhBMP-2.

Effect of rhBMP-2 and rhBMP-9 on Cell Adhesion and Proliferation

As shown in Fig. 2, 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

proliferation at 1 , 3 and 5 days post-seeding with the exception that rhBMP-9 high group demonstrated significantly lower cell proliferation at 3 days when compared to control samples.

Effect of rhBMP-2 and rhBMP-9 on Osteoblast Differentiation

As shown in 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). Interestingly, only 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). Furthermore, 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).

As can be seen in Fig. 1 , 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).

The effect of rhBMP-2 and rhBMP-9 had the greatest influence on osteoblast differentiation. Previously, it has been shown that rhBMP-2 in combination with a collagen membrane was able to significantly upregulate the differentiation of osteoblasts in vitro ((Miron et al., 2013). Here it could be show that 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). Moreover, both concentrations of rhBMP-9 induced a 3-fold and thus clearly significant increase in alizarin red staining when compared to rhBMP-2 groups at 14 days (Fig. 5). Thus, collagen membranes combined with rhBMP-9 have a stronger osteopromotive potential than when combined with rhBMP-2.

Notably, at 14 days post seeding, 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).

Effect of rhBMP2 and rhBMP9 on cell adhesion and proliferation

Following pre-coating NBM (natural bone mineral) particles with rhBMP-2 and rhBMP-9 at the various concentrations, the effects of cell attachment was

investigated 8 hours post-seeding (results not shown). It was found that all cells attached to NBM particles in similar fashion with no significant differences observed between groups (results not shown). Thereafter, cell proliferation was determined at 1 , 3 and 5 days post seeding (results not shown). Under the provided in vitro conditions, it was found that rhBMP-2 low and high as well as rhBMP-9 high stimulated significantly higher cell numbers at 3 days post seeding when compared to control samples (results not shown). No significant difference could be observed between all treatments groups at 5 days post seeding (results not shown).

Effect of rhBMP-2 and rhBMP-9 on osteoblast differentiation

The effects of both tested concentrations of rhBMPs on osteoblast cell differentiation were then investigated via real-time PCR, ALP staining and alizarin red staining (see, e.g, Figs. 7-9). ALP staining was first performed to investigate the effects of rhBMP-2 and rhBMP-9 on early osteoblast differentiation (Figs. 7, 8). It could be shown that rhBMP-9 at both concentrations led to significantly and markedly higher ALP staining when compared to rhBMP-2 by demonstrating more than two, 3.4, 5, 6, 7, 8, 9, 10, 15, 20 times and in fact up to 25 times higher ALP staining when combined with NBM particles (Fig. 8). Interestingly, observation of particles following 7 days revealed the presence of ALP staining on the grafting material surfaces in web-like form when bone grafts were coated with rhBMP-9 at either low or high concentrations (Fig. 7). It was also found that all concentrations of either rhBMP-2 or rhBMP-9 significantly upregulated Runx2, Col1 a2 and OCN when compared to control samples (Fig.9 A, B, D). Interestingly, only rhBMP-9 was able to significantly increase ALP mRNA levels approximately 8 fold at 3 days when compared to all other modalities. By 14 days, no significant differences in any of the investigated genes could be observed between treatment groups (Fig. 9). Alizarin red staining revealed higher and more densely stained particles (Fig.10) on rhBMP-9 coated NBM particles with color thresholding demonstrating significantly higher levels found on rhBMP-9 high particles when compared to control and rhBMP-2 low samples (results not shown).

MATERIALS AND METHODS:

IN VITRO STUDIES

Regents and cell lines

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) and rhBMP-9 (CHO-derived Ser320-Arg429, Accession no. Q9UK05, Cat. No. 3209-BP as of Jan. 2016) were purchased from R&D Systems Inc. (Minneapolis, MM, USA).

For in vitro experiments shown in Figs. 1 -5, the following 5 groups, 1 ) control;

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

INVITROGEN, INC.), 10% fetal bovine serum (FBS, INVITROGEN INC.), and antibiotics (INVITROGEN, INC.). Osteogenic medium containing growth medium with 50 μg/ml ascorbic acid (SIGMA, USA) and 10 mM β-glycerophosphate (SIGMA, USA) were used for real-time PCR and alizarin red staining to promote osteoblast differentiation as previously described (Miron et al., 201 1 ). A 10 mm x 10 mm sized membrane was placed at the bottom of 24 well dishes and coated with rhBMP-2 or - 9 for 5 minutes prior to cell seeding. Thereafter cells were seeded onto the various treatment modalities at a density of 10,000 cells in 24 well culture plates for cell migration, adhesion and proliferation experiments and 50,000 cells per well in 24 well dishes for real-time PCR, and alizarin red experiments. For experiments lasting longer than 5 days, medium was replaced twice weekly.

For all in vitro experiments based on NBM including those discussed but not shown, the following 5 groups were used 1 ) control; a natural bone mineral (NBM) bone grafting material (BIO-OSS, Geistlich, Switzerland) alone, 2) low concentration (10 ng/ml) of rhBMP2 with NBM, 3) high concentration (100 ng/ml) of rhBMP2 with NBM, 4) low concentration (10 ng/ml) of rhBMP9 with NBM, and 5) high

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.). For in vitro experiments, 8 mg of NBM was placed at the bottom of 24 well dishes and coated with rhBMP-2 or -9 for 5 minutes prior to cell seeding. Thereafter cells were seeded onto the various treatment modalities at a density of 10,000 cells in 24 well culture plates for cell adhesion and proliferation experiments and 50,000 cells per well in 24 well dishes for real-time PCR, ALP assay and alizarin red experiments. For experiments lasting longer than 5 days, medium was replaced twice weekly.

Adhesion and Proliferation Assay

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). At desired time points, cells were washed with phosphate buffered solution (PBS) and quantified using a fluorescence plate reader ELx808 Absorbance reader, BIO-TEK). Real-time PCR analysis for osteoblast differentiation markers

Total RNA was isolated using High Pure RNA Isolation Kit (Roche, Switzerland) at 3 and 14 days for osteoblast differentiation markers. 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.

MINERALIZATION ASSAYS

Alizarin red staining

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 μg/ml ascorbic acid (Sigma, St. Louis, MO) and 10 mM β- 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. Louis, MO, USA) in water (pH 6.4) at room temperature for 1 hour as previously described (Miron et al., 201 1 ). Alizarin red staining was performed using images captured on a microscope (Wild Heerbrugg M400 ZOOM Makroskop). Image J software was used to quantify data using set parameters for color intensity staining of red using color threshold including parameters for hue, saturation and brightness. The same threshold values were used for all analyzed.

ALP (alkaline phosphatase) activity assay

After a number of days, cells were quantified for alkaline phosphatase expression as determined by cell imaging. 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. Following 2 min of rinsing in deionized water. All images were captured on a Wild Heerbrugg M400 ZOOM Makroskop (WILD HEERBRUGG, Switzerland) at the same magnification at the same light intensity and imported onto Image J software. Thresholding was used to generate percent stained values for each field of view.

Statistical Analysis

All experiments were performed in triplicate with three independent experiments for each condition. Data were analyzed for statistical significance using a one-way or two-way analysis of variance with Bonferroni test (^*, p values < 0.05 was considered significant) by GraphPad Prism 6.0 software.

IN VIVO STUDIES

Animals

48 adult, female New Zealand rabbits weighing between 3 and 4 kg were used. All animal experiments were approved by the Committee for Animal Research, State of Bern, Switzerland. Animals were housed in the Central Animal Facility of the University of Bern with an adjusted climate (temperature 22-24^°C±2^°C, humidity 30- 60% ±5%, a light:dark cycle of 12:12 hours), without excessive or startling noises and with standard diet and water ad libitum.

Surgical procedure

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. In the area of the right and left parietal and frontal bones, 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,

Switzerland) (iii) BO + rhBMP2_5 g (iv) BO + rhBMP2_20 g (v) BO + rhBMP9_5 g, and (vi) BO + rhBMP9_20 g. 20mg of BO mixed with 10μΙ of rhBMP solution was filled in each defect and the defects were covered with BioGide® (Geistlich Pharma AG) in 8 mm diameter cut using biopsy punch. After carefully filling the defects, the periosteum and skin were closed with interrupted sutures in layers using 3-0 Vicryl and 4-0 Nylon sutures. After a healing period of 8 weeks, the rabbits were sacrificed by an overdose of ketamin. The skull containing all four craniotomy sites was removed and placed in 10% neutral formalin.

Micro-CT analysis

The 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,

Bruettisellen, Switzerland). 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,

Dusseldorf, Germany). The region of interest (ROI) 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.

EXAMPLES OF MATRIXES/SUBRATES

TISSEEL fibrin sealant:

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:

Under the trademark 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

chloride 6.9 mg, water.

A collagen sponge utilized during routine extraction socket healing

(Collacone® by Botiss):

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.

After tooth removal, the healing of an extraction socket requires the formation and maturation of a blood clot, followed by the infiltration of fibroblasts that replace the coagulum; finally, the application of a provisional matrix allows the formation of new bone tissue. The spongy structure of Collacone® ensures an easy and fast application in extraction sockets. Its combination with rhBMP-9 induces new bone formation in patients requiring tooth extraction.

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 deproteinized bovine derived bone mineral (BioOss®, Geistlich):

Geistlich Bio-Oss® is one of the leading bone substitute for regenerative dentistry worldwide. The osteoconductive properties of Geistlich Bio-Oss® lead to effective and predictable bone regeneration. Particles are incorporated over time within living bone which provides long-term volume preservation. The biofunctionality of Geistlich Bio-Oss® is characterized by its topographic structure, hydrophilic properties that supports reliable bone formation. These bone grafts were utilized in particles up to 1 mm in size.

A demineralized freeze-dried allograft from human origin (DBX):

Synthes is the leader in orthopaedic trauma devices for internal and external fixation. 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 (β-TCP). 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 μηι) 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.

An injectable synthetic paste containing bone particles (MAXRESORB Inject, Botiss):

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 Membranes (Botiss Biomaterials):

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. Based on these advantageous properties, 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 (Botiss Biomaterials):

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.

PCR primers for genes encoding Runx2, ALP, COL1a2, OCN and

Gene Primer Sequence

mRUNX2 F Agggactatggcgtcaaaca [SEQ ID NO: 2]

mRUNX2 R Ggctcacgtcgctcatctt [SEQ ID NO: 3]

mOCN F Cagacaccatgaggaccatc [[SEQ ID NO: 4]

mOCN R Ggactgaggctctgtgaggt [SEQ ID NO: 5]

mALP F Ggacaggacacacacacaca [SEQ ID NO: 6]

mALP R Caaacaggagagccacttca [SEQ ID NO: 7]

mCOLa2 F Gagctggtgtaatgggtcct [SEQ ID NO: 8] mCOLa2 R Gagacccaggaagacctctg [SEQ ID NO: 9] mGAPDH F Aggtcggtgtgaacggatttg [SEQ ID NO: 10]

mGAPDH R Tgtagaccatgtagttgaggtca [SEQ ID NO: 1 1 ]

The invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed, since these embodiments are intended as illustrations of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

Various references are cited herein, the disclosures of which are incorporated by reference in their entireties.

LIST OF REFERENCES

Boden et al., The use of rhBMP-2 in interbody fusion cages. Definitive evidence of osteoinduction in humans: a preliminary report, Spine 2000 Feb 1 ; 25(3): 376-381 .

Franceschi et al., Gene therapy for bone formation: in vitro and in vivo osteogenic activity of an adenovirus expressing BMP7, . Cell Biochem. 2000 Jun 6; 78(3): 476- 486.

Govender et al., Recombinant Human Bone Morphogenetic Protein-2 for Treatment of Open Tribial Fractures, Bone Joint Surg. Am. 2002 Dec; 84 (12): 2123-2134.

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.

Miron et al., Osteogenic potential of autogenous bone grafts harvested with four different surgical techniques"; J Dent Res 201 1 ;90:1428-1433.

Tannoury et al., Complications with the use of bone morphogenetic protein 2 (BMP- 2) in spine surgery, Spine J. 2014 Mar 1 ; 14(3): 552-559.

Van de Watering FC, 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.

SEQUENCE LISTING

<210> 1

<211> 429

<212> PRT

<213> Homo sapiens

<400> 1

Met Cys Pro Gly Ala Leu Trp Val Ala Leu Pro Leu Leu Ser Leu Leu 1 5 10 15

Ala Gly Ser Leu Gin Gly Lys Pro Leu Gin Ser Trp Gly Arg Gly Ser

20 25 30

Ala Gly Gly Asn Ala His Ser Pro Leu Gly Val Pro Gly Gly Gly Leu

35 40 45

Pro Glu His Thr Phe Asn Leu Lys Met Phe Leu Glu Asn Val Lys Val 50 55 60

Asp Phe Leu Arg Ser Leu Asn Leu Ser Gly Val Pro Ser Gin Asp Lys 65 70 75 80

Thr Arg Val Glu Pro Pro Gin Tyr Met lie Asp Leu Tyr Asn Arg Tyr

85 90 95

Thr Ser Asp Lys Ser Thr Thr Pro Ala Ser Asn lie Val Arg Ser Phe

100 105 110

Ser Met Glu Asp Ala lie Ser lie Thr Ala Thr Glu Asp Phe Pro Phe

115 120 125

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

Asp Pro Ser His Asp Leu Lys Gly Ser Val Val lie Tyr Asp Val Leu

165 170 175

Asp Gly Thr Asp Ala Trp Asp Ser Ala Thr Glu Thr Lys Thr Phe Leu

180 185 190 Val Ser Gin Asp lie Gin Asp Glu Gly Trp Glu Thr Leu Glu Val Ser 195 200 205

Ser Ala Val Lys Arg Trp Val Arg Ser Asp Ser Thr Lys Ser Lys Asn 210 215 220

Lys Leu Glu Val Thr Val Glu Ser His Arg Lys Gly Cys Asp Thr Leu 225 230 235 240

Asp lie Ser Val Pro Pro Gly Ser Arg Asn Leu Pro Phe Phe Val Val

245 250 255

Phe Ser Asn Asp His Ser Ser Gly Thr Lys Glu Thr Arg Leu Glu Leu

260 265 270

Arg Glu Met lie Ser His Glu Gin Glu Ser Val Leu Lys Lys Leu Ser

275 280 285

Lys Asp Gly Ser Thr Glu Ala Gly Glu Ser Ser His Glu Glu Asp Thr 290 295 300

Asp Gly His Val Ala Ala Gly Ser Thr Leu Ala Arg Arg Lys Arg Ser 305 310 315 320

Ala Gly Ala Gly Ser His Cys Gin Lys Thr Ser Leu Arg Val Asn Phe

325 330 335

Glu Asp lie Gly Trp Asp Ser Trp lie lie Ala Pro Lys Glu Tyr Glu

340 345 350

Ala Tyr Glu Cys Lys Gly Gly Cys Phe Phe Pro Leu Ala Asp Asp Val

355 360 365

Thr Pro Thr Lys His Ala lie Val Gin Thr Leu Val His Leu Lys Phe 370 375 380

Pro Thr Lys Val Gly Lys Ala Cys Cys Val Pro Thr Lys Leu Ser Pro 385 390 395 400 lie Ser Val Leu Tyr Lys Asp Asp Met Gly Val Pro Thr Leu Lys Tyr

405 410 415 Tyr Glu Gly Met Ser Val Ala Glu Cys Gly Cys Arg 420 425

<210> 2

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> PCR primer

<400> 2

agggactatg gcgtcaaaca

20

<210> 3

<211> 19

<212> DNA

<213> Artificial Sequence

<220>

<223> PCR primer

<400> 3

ggctcacgtc gctcatctt

19

<210> 4

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> PCR primer

<400> 4

cagacaccat gaggaccatc

20

<210> 5

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> PCR primer

<400> 5

ggactgaggc tctgtgaggt

20 <210> 6

<211> 20

<212> DNA

<213> Artificial Sequence <220>

<223> PCR primer

<400> 6

ggacaggaca cacacacaca 20

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence <220>

<223> PCR primer

<400> 7

caaacaggag agccacttca 20

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence <220>

<223> PCR primer

<400> 8

gagctggtgt aatgggtcct 20

<210> 9

<211> 20

<212> DNA

<213> Artificial Sequence <220>

<223> PCR primer

<400> 9

gagacccagg aagacctctg 20

<210>

<211> <212> DNA

<213> Artificial Sequence <220>

<223> PCR primer

<400> 10

aggtcggtgt gaacggattt g 21

<210> 11

<211> 23

<212> DNA

<213> Artificial Sequence <220>

<223> PCR primer

<400> 11

tgtagaccat gtagttgagg tea 23

Claims

WHAT WE CLAIM IS:

1 . A method for promoting bone growth and/or regeneration comprising:

administering to a human patient in need thereof:

(c) at least one substrate and or matrix, and

(d) 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).

2. The method of claim 1 , wherein said substrate is an implant such as a dental implant, a surgical implant including a plastic surgical implant or an orthopedic implant.

3. The method of claim 1 or 2, wherein said substrate comprises ceramics, titanium, collagen and zirconium.

4. The method of any one of the preceding claims, wherein the rhBMP-9 coats at least a part of said substrate or matrix.

5. The method of any one of the preceding claims, wherein the rhBMP-9 is applied intra-osseal.

6. The method of any one of the preceding claims, wherein said promoting bone growth and/or regeneration comprises differentiation into osteoblasts, proliferation of osteoblasts and/or adhesion of osteoblasts to the substrate or matrix.

7. The method of claim 6, wherein promoting bone growth and/or regeneration comprises differentiation into osteoblasts.

8. The method of claim any one of the preceding claims, wherein the patient suffers from osteoporosis or suffered bone loss.

9. The method of any one of the preceding claims, further comprising 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.

10. A composition or kit for promoting bone growth and/or inhibiting bone loss comprising:

(a) a rhBMP-9,

wherein the rhBMP-9 comprises amino acids 320 to 429 of SEQ ID NO: 1 or has 80%, 90%, 95, 98, 99% or complete sequence identify with amino acids 318 to 429 of SEQ ID NO: 1 , and/or

wherein the rhBMP-9 comprises at least 80, 90, 100 consecutive amino acids of amino acids 320 to 429 of SEQ ID NO: 1 ; or

(b) a amino acid sequence comprising:

- amino acids 320 to 429 of SEQ ID NO: 1 or a sequence having 80%, 90%, 95, 98, 99% or complete sequence identify with amino acids 320 to 429 of SEQ ID NO: 1 , or

at least 80, 90, 100 consecutive amino acids of amino acids 320 to 429 of SEQ ID NO: 1 ; and

(b) one or more three- dimensional substrates or matrixes, wherein the

rhBMP-9 of (a) or amino acid of (b) optionally comprises one or more posttranslational modifications such as glycosylations.

1 1 . The kit of claim 10, wherein (a) and (b) are provided in separate containers and include instructions for using (a) and (b) together for promoting said bone growth and/or inhibiting said bone loss.

12. The composition of claim 10 or kit according to claims 10 or 1 1 , wherein said posttranslational modification is a glycosylation, preferably conferred by expression in an eukaryotic cell.

13. The composition or kit of claim 10 or any one of the subsequent claims, wherein said one or more substrates or matrixes comprises a membrane or a sponge.

14. The composition or kit of claim 10 or any one of the subsequent claims, wherein the one or more substrates or matrixes are one or more of the following bone graft 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.

15. The composition or kit of claim 10 or any one of the subsequent claims

wherein the one or more matrixes are:

- at least one liquid matrix comprising 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.

16. The composition or kit of claim 10 or any one of the subsequent claims, wherein the one or more substrates or matrixes comprise cross-linked or non-cross- linked collagen, collagen bi-products comprising synthetic or natural bone material, e-PTFE, d-PTFE, or combinations thereof.

17. The composition or kit of claim 10 or any one of the subsequent claims, wherein the one or more substrates or matrixes comprise cross-linked or non-cross- linked collagen and one or more bone graft materials, wherein said collagen preferably covers said bone graft material.

18. The composition or kit of claim 10 or any one of the subsequent claims, wherein said one or more substrates or matrixes comprise calcium phosphates, calcium sulfurs, hydroxyapatite and derivatives thereof, biphasic calcium phosphates, orthophosphates, beta-tricalcium phosphates, alpha-tricalcium phosphates or combinations thereof.

19. The composition or kit claim 10 or any one of the subsequent claims, wherein said one or more substrates or matrixes comprise polyesters such as poly(glycolic acid) (PGA), poly(lactic acid) (PLA), poly (ε-caprolactone) (PCL), or co-polymers and/or combinations thereof.

20. The composition or kit of claim 10 or any one of the subsequent claims, further comprising 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.

21 . The composition or kit of claim 10 or any one of the subsequent claims, wherein the rhBMP-9 or the amino acid sequence covers the substrate or matrix, wherein the matrix and/or the rhBMP-9 or part thereof is applied via 3-D printing technologies.

22. The method of any one of claims of 1 to 10, wherein the rhBMP-9 or the amino acid sequence is part of any one of the compositions or kits of claims 1 1 to 21 .