CA1341610C - Osteogenic polypeptides - Google Patents

Abstract

Disclosed are 1) osteogenic devices comprising a matrix containing osteogenic protein and methods of inducing endochondral bone growth in mammals using the devices; 2) amino acid sequence data, amino acid composition, solubility properties, structural features, homologies and various other data characterizing osteogenic proteins, and 3) methods of producing osteogenic proteins using recombinant DNA technology.

Description

This invention relates to osteogenic devices, to genes encoding proteins which can induce osteogenesis in mammals and methods for their production using recombinant DNA techniques, to a method of reproducibly purifying osteogenic protein from mammalian bone, and to bone and cartilage repair procedures using the osteogenic device.

Mammalian bone tissue is known to contain, one or more proteinaceous materials, presumably active during growth and natural bone healing, which can induce a developmental cascade of cellular events resulting in endochondral bone formation. This active factor (or factors) has variously been referred to in the literature as bone morphogenetic or morphogenic protein, bone inductive protein, osteogenic protein, osteogenin, or osteoinductive protein.

The developmental cascade of bone differentiation consists of recruitment of mesenchymal cells, proliferation of progenitor cells, calcification of cartilage, vascular invasion, bone formation, remodeling, and finally marrow differentiation (Reddi (1981) Collagen Rel. Res.
1.209-226).

1 Though the precise mechanisms underlying these phenotypic transformations are unclear, it has been shown that the natural endochondral bone differentiation activity of bone matrix can be dissociatively extracted and reconstituted with inactive residual collagenous matrix to restore full bone induction activity (Sampath and Reddi, (1981) Proc. Natl. Acad. Sci. USA 71:7599-7603). This provides an experimental method for assaying protein extracts for their ability to induce endochondral bone in vivo.

This putative bone inductive protein has been shown to have a molecular mass of less than 50 kilodaltons (kD). Several species of mammals produce closely related protein as demonstrated by cross species implant experiments (Sampath and Reddi (1983) Proc. Natl. Acad. Sci. USA 80:6591-6595).

The potential utility of these proteins has been widely recognized. It is contemplated that the availability of the protein would revolutionize orthopedic medicine, certain types of plastic surgery, and various periodontal and craniofacial reconstructive procedures.

The observed properties of these protein fractions have induced an intense research effort in various laboratories directed to isolating and identifying the pure factor or factors responsible for osteogenic activity. The current state of the art of purification of osteogenic protein from mammalian bone is disclosed by Sampath et al. (Proc.
Natl. Acad. Sci. USA (1987) J,Q). Urist et al. (Proc.
Soc. Exp. Biol. Med. (1984) 1:194-199) disclose a human osteogenic protein fraction which was extracted from demineralized cortical bone by means of a calcium chloride-urea inorganic-organic solvent mixture, and retrieved by differential precipitation in guanidine-hydrochloride and preparative gel electrophoresis. The authors report that the protein 1(1 fraction has an amino acid composition of an acidic polypeptide and a molecular weight in a range of 17-18 kD.

Urist et al. (Proc. Natl. Acad. Sci. USA
(1984) 81:371-375) disclose a bovine bone morphogenetic protein extract having the properties of an acidic polypeptide and a molecular weight of approximately 18 kD. The authors reported that the protein was present in a fraction separated by hydroxyapatite chromatography, and that it induced bone formation in mouse hindquarter muscle and bone regeneration in trephine defects in rat and dog skulls. Their method of obtaining the extract from bone results in ill-defined and impure preparations.
European Patent Application Serial No.
148,155, published October 7, 1985, purports to disclose osteogenic proteins derived from bovine, porcine, and human origin. One of the proteins, designated by the inventors as a P3 protein having a molecular weight of 22-24 kD, is said to have been 1 purified to an essentially homogeneous state. This material is reported to induce bone formation when implanted into animals.

International Publication No. WO 88/00205, published January 14, 1988, discloses an impure fraction from bovine bone which has bone induction qualities. The named applicants also disclose putative bone inductive factors produced by recombinant DNA techniques. Four DNA sequences were retrieved from human or bovine genomic or cDNA
libraries and apparently expressed in recombinant host cells. While the applicants stated that the expressed proteins may be bone morphogenic proteins, bone induction was not demonstrated, suggesting that the recombinant proteins are not osteogenic. See also Urist et al., EP 0,212,474 entitled Bone Morphogenic Agents.

Wang et al. (Proc. Nat. Acad. Sci. USA
(1988) $a: 9484-9488) discloses the purification of a bovine bone morphogenetic protein from guanidine extracts of demineralized bone having cartilage and bone formation activity as a basic protein corresponding to a molecular weight of 30 kD
determined from gel elution. Purification of the protein yielded proteins of 30, 18 and 16 kD which, upon separation, were inactive. In view of this result, the authors acknowledged that the exact identity of the active material had not been determined.

1 _ -Wozney et al. (Science (1988) 242:
1528-1534) discloses the isolation of full-length cDNA's encoding the human equivalents of three polypeptides originally purified from bovine bone.

5 The authors report that each of the three recombinantly expressed human proteins are independently or in combination capable of inducing cartilage formation. No evidence of bone formation is reported.
in It is an object of this invention to provide osteogenic devices comprising matrices containing dispersed osteogenic protein capable of bone induction in allogenic and xenogenic implants.
Another object is to provide a reproducible method of isolating osteogenic protein from mammalian bone tissue. Another object is to characterize the protein responsible for osteogenesis. Another object is to provide natural and recombinant osteogenic proteins capable of inducing endochondral bone formation in mammals, including humans. Yet another object is to provide genes encoding osteogenic proteins and methods for their production using recombinant DNA techniques. Another object is to provide methods for inducing cartilage formation.
These and other objects and features of the invention will be apparent from the description, drawings, and claims which follow.

1 Summary of the Invention This invention involves osteogenic devices which, when implanted in a mammalian body, can induce at the locus of the implant the full developmental cascade of endochondral bone formation and bone marrow differentiation. Suitably modified as disclosed herein, the devices also may be used to induce cartilage formation. The devices comprise a carrier material, referred to herein as a matrix, having the characteristics disclosed below, containing dispersed osteogenic protein either in its native form as purified from natural sources or produced using recombinant DNA techniques.

A key to these developments was the elucidation of amino acid sequence and structure data of native osteogenic protein. A protocol was developed which results in retrieval of active, substantially pure osteogenic protein from mammalian bone. The protein has a half-maximum bone forming activity of about 0.8 to 1.0 ng per mg of implant.
The proteins are believed to dimerize during refolding. They appear not to be active when reduced. Various combinations of species of the proteins, i.e., heterodimers, have activity, as do homodimers.

The invention provides native forms of osteogenic protein, extracted from bone or produced using recombinant DNA techniques. The substantially 1 pure osteogenic protein may include forms having varying glycosylation patterns, varying N-termini, a family of related proteins having regions of amino acid sequence homology, and active truncated or mutated forms of native protein, no matter how derived. The naturally sourced osteogenic protein in its native form is glycosylated and has an apparent molecular weight of about 30 kD as determined by SDS-PAGE. When reduced, the 30 kD protein gives rise to two glycosylated polypeptide chains having apparent molecular weights of about 16 kD and 18 kD.
In the reduced state, the 30 kD protein has no detectable osteogenic activity. The deglycosylated protein, which has osteogenic activity, has an apparent molecular weight of about 27 kD. When reduced, the 27 kD protein gives rise to the two deglycosylated polypeptides have molecular weights of about 14 kD to 16 kD.

Analysis of digestion fragments indicate that the native 30 kD osteogenic protein contains the following amino acid sequences (question marks indicate undetermined residues):

(1) S-F-D-A-Y-Y-C-S-G-A-C-Q-F-P-M-P-K;
(2) S-L-K-P-S-N-Y-A-T-I-Q-S-I-V;
(3) A-C-C-V-P-T-E-L-S-A-I-S-M-L-Y-L-D-E-N-E-K;
(4) M-S-S-L-S-I-L-F-F-D-E-N-K;
(5) S-Q-E-L-Y-V-D-F-Q-R;
(6) F-L-H-C-Q-F-S-E-R-N-S;
(7) T-V-G-Q-L-N-E-Q-S-S-E-P-N-I-Y;

1 (8) L-Y-D-P-M-V-V;

(9) V-G-V-V-P-G-I-P-E-P-C-C-V-P-E;

(10) V-D-F-A-D-I-G;

(11) V-P-K-P-C-C-A-P-T;

(12) I-N-I-A-N-Y-L;

(13) D-N-H-V-L-T-M-F-P-I-A-I-N;

(14) D-E-Q-T-L-K-K-A-R-R-K-Q-W-I-?-P;

(15) D-I-G-?-S-E-W-I-I-?-P;

(16) S-I-V-R-A-V-G-V-P-G-I-P-E-P-?-?-V;

(17) D-?-I-V-A-P-P-Q-Y-H-A-F-Y;

(18) D-E-N-K-N-V-V-L-K-V-Y-P-N-M-T-V-E;

(19) S-Q-T-L-Q-F-D-E-Q-T-L-K-?-A-R-?-K-Q;

(20) D-E-Q-T-L-K-K-A-R-R-K-Q-W-I-E-P-R-N-?-A-R-R-Y-L;

(21) A-R-R-K-Q-W-I-E-P-R-N-?-A-?-R-Y-?-?-V-D; and (22) R-?-Q-W-I-E-P-?-N-?-A-?-?-Y-L-K-V-D-?-A-?-?-G.
The availability of the protein in substantially pure form, and knowledge of its amino acid sequence and other structural features, enable the identification, cloning, and expression of native genes which encode osteogenic proteins. When properly modified after translation, incorporated in a suitable matrix, and implanted as disclosed herein, these proteins are operative to induce formation of cartilage and endochondral bone.

Consensus DNA sequences designed as disclosed herein based on partial sequence data and observed homologies with regulatory proteins disclosed in the literature are useful as probes for extracting genes encoding osteogenic protein from genomic and cDNA libraries. One of the consensus _9_ 1341610 1 sequences has been used to isolate a heretofore unidentified genomic DNA sequence, portions of which when ligated encode a protein having a region capable of inducing endochondral bone formation. This protein, designated OP1, has an active region having the sequence set forth below.

YMNATN--H-AIVQTLVHFINPET-VPKPCCAPTQLNA

ISVLYFDDSSNVILKKYRNMVVRACGCH
A longer active sequence is:

HQRQA

'50 60 70 YMNATN--H-AIVQTLVHFINPET-VPKPCCAPTQLNA

ISVLYFDDSSNVILKKYRNMVVRACGCH
Fig. 1A discloses the genomic DNA sequence of OP1.

The probes have also retrieved the DNA sequences identified.in WO 88/00205, referenced above, designated therein as BMPII(b) and BMPIII. The inventors herein have discovered that certain subparts of these genomic DNAs, and BMPIIa, from the same publication, when properly assembled, encode proteins (CBMPIIa, CBMPIIb, and CBMPIII) which have true osteogenic activity, i.e., induce the full cascade of events when properly implanted in a mammal 1 leading to endochondral bone formation. These sequences are:

CBMP-2a CKRHPLYVDFS-DVGWNDWIVAPPGYHAFYCHGECPFPLAD

HLNSTN--H-AIVQTLVNSVNS-K-IPKACCVPTELSA

ISMLYLDENEKVVLKNYQDMVVEGCGCR

CBMP-2b CRRHSLYVDFS-DVGWNDWIVAPPGYQAFYCHGDCPFPLAD

HLNSTN--H-AIVQTLVNSVNS-S-IPKACCVPTELSA

ISMLYLDEYDKVVLKNYQEMVVEGCGCR

SLKPSN--H-ATIQSIVRAVGVVPGIPEPCCVPEKMSS

LS ILFFDENKNVVLKVYPNMTVESCACR

Thus, in view of this disclosure, skilled genetic engineers can isolate genes from cDNA or genomic libraries which encode appropriate amino acid sequences, and then can express them in various types of host cells, including both procaryotes and eucaryotes, to produce large quantities of active proteins capable of inducing bone formation in mammals including humans.

The substantially pure osteogenic proteins (i.e., proteins free of contaminating proteins having.no osteoinductive activity) are useful in clinical applications in conjunction with a suitable delivery or support system (matrix). The matrix is made up of particles or 1 porous materials. The pores must be of a dimension to permit progenitor cell migration and subsequent differentiation and proliferation. The particle size should be within the range of 70 -850 pm, preferably 70 - 420 pm. It may be fabricated by close packing particulate material into a shape spanning the bone defect, or by otherwise structuring as desired a material that is biocompatible (non-inflammatory) and, biodegradable in vivo to serve as a "temporary scaffold" and substratum for recruitment of migratory progenitor cells, and as a base for their subsequent anchoring and proliferation.
Currently preferred carriers include particulate, demineralized, guanidine extracted, species-specific (allogenic) bone, and particulate, deglycosglated, protein extracted, demineralized, xenogenic bone. Optionally, such zenogenic bone powder matrices also may be treated with proteases such as trypsin. Other useful matrix materials comprise collagen, homopolymers and copolymers of glycolic acid and lactic acid, hydroxyapatite, tricalcium phosphate and other calcium phosphates.

The osteogenic proteins and implantable osteogenic devices enabled and disclosed herein will permit the physician to obtain optimal predictable bone formation to correct, for example, acquired and congenital craniofacial and I r 1341610 1 other skeletal or dental anomalies (Glowacki et al. (1981) Lancet 1:959-963). The devices may be used to induce local endochondral bone formation in non-union fractures as demonstrated in animal tests, and in other clinical applications including periodontal applications where bone formation is required. Another potential clinical application is in cartilage repair, for example, in the treatment of osteoarthritis.

- -1 Brief Description of the Drawing The foregoing and other objects of this invention, the various features thereof, as well as the invention itself, may be more fully understood from the following description, when read together with the accompanying drawings, in which:

FIGURE 1A represents the nucleotide sequence of the genomic copy of osteogenic protein "OP1"
gene. The unknown region between 1880 and 1920 actually represents about 1000 nucleotides;
FIGURE 1B is a representation of the hybridization of the consensus gene/probe to the osteogenic protein "OP1" gene;

FIGURE 2 is a collection of plots of protein concentration (as indicated by optical absorption) vs elution volume illustrating the results of bovine osteogenic protein (BOP) fractionation during purification on heparin-Sepharose-I; HAP-Ultragel;
sieving gel (Sephacryl*300); and heparin-Sepharose-II;

FIGURE 3 is a photographic reproduction of a Coomassie blue stained SDS polyacrylamide gel of the osteogenic protein under non-reducing (A) and reducing (B) conditions;

FIGURE 4 is a photographic reproduction of a Con A blot of an SDS polyacrylamide gel showing the carbohydrate component of oxidized (A) and reduced (B) 30 kD protein;

* Trade-mark 1 FIGURE 5 is a photographic reproduction of an autoradiogram of an SDS polyacrylamide gel of 125I-labelled glycosylated and deglycosylated osteogenic protein under non-reducing (A) and reducing (B) conditions;

FIGURE 6 is a photographic reproduction of an autoradiogram of an SDS polyacrylamide gel of peptides produced upon the digestion of the 30 kD
osteogenic protein with V-8 protease (B), Endo Lys C
protease (C), pepsin (D), and trypsin (E). (A) is control;

FIGURE 7 is a collection of HPLC
chromatograms of tryptic peptide digestions of 30 kD
BOP (A), the 16 kD subunit (B), and the 18 kD subunit (C);

FIGURE 8 is an HPLC chromatogram of an elution profile on reverse phase C-18 HPLC of the samples recovered from the second heparin-Sepharose chromatography step (see FIGURE 2D). Superimposed is the percent bone formation in each fraction;

FIGURE 9 is a gel permeation chromatogram of an elution profile on TSK 3000/2000 gel of the C-18 purified osteogenic peak fraction. Superimposed is the percent bone formation in each fraction;

FIGURE 10 is a collection of graphs of protein concentration (as indicated by optical absorption) vs. elution volume illustrating the results of human protein fractionation on A

1 heparin-Sepharose I (A), HAP-Ultragel (B), TSK
3000/2000 (C), and heparin-Sepharose II (D). Arrows indicate buffer changes;

FIGURE 11 is a graph showing representative dose response curves for bone-inducing activity in samples from various purification steps including reverse phase HPLC on C-18 (A), Heparin-Sepharose II
(B), Sephacryl S-300 HR (C), HAP-ultragel (D), and Heparin-Sepharose I (E);

FIGURE 12 is a bar graph of radiomorphometric analyses of feline bone defect repair after treatment with an osteogenic device (A), carrier control (B), and demineralized bone (C);
FIGURE 13 is a schematic representation of the DNA sequence and corresponding amino acid sequence of a consensus gene/probe for osteogenic protein (COPO);

FIGURE 14 is a graph of osteogenic activity vs. increasing molecular weight showing peak bone forming activity in the 30 kD region of an SDS
polyacrylamide gel;

FIGURE 15 is a photographic representation of a Coomassie blue stained SDS gel showing gel purified subunits of the 30 kD protein;

FIGURE 16 is a pair of HPLC chromatograms of Endo Asp N proteinase digests of the 18 kD subunit (A) and the 16 kD subunit (B);

1 FIGURE 17 is a photographic representation of the histological examination of bone implants in the rat model: carrier alone (A); carrier and glycosylated osteogenic protein (B); and carrier and deglycosylated osteogenic protein (C). Arrows indicate osteoblasts;

FIGURE 18 is a graph illustrating the activity of xenogenic matrix (deglycolylated bovine matrix);

FIGURES 19A and 19B are bar graphs showing the specific activity of naturally sourced OP before and after gel elution as measured by calcium content vs. increasing concentrations of proteins (dose curve, in ng);.

1 Description Purification protocols have been developed which enable isolation of the osteogenic protein present in crude protein extracts from mammalian bone. While each of the separation steps constitute a known separation technique, it has been discovered that the combination of a sequence of separations exploiting the protein's affinity for heparin and for hydroxyapatite (HAP) in the presence of a denaturant such as urea is key to isolating the pure protein from the crude extract. These critical separation steps are combined with separations on hydrophobic media, gel exclusion chromatography, and elution form SDS PAGE.

The isolation procedure enables the production of significant quantities of substantially pure osteogenic protein from any mammalian species, provided sufficient amounts of fresh bone from the species is available. The empirical development of the procedure, coupled with the availability of fresh calf bone, has enabled isolation of substantially pure bovine osteogenic protein (BOP). BOP has been characterized significantly as set forth below; its ability to induce cartilage and ultimately endochondral bone growth in cat, rabbit, and rat have been studied; it has been shown to be able to induce the full developmental cascade of bone formation previously ascribed to unknown protein or proteins in heterogeneous bone extracts; and it may be used to induce formation of endochondral bone in orthopedic defects including non-union fractures. In its native form it is a glycosylated, dimeric protein. However, it is active in deglycosylated form. It has been partially sequenced. Its primary structure includes the amino acid sequences set forth herein.

Elucidation of the amino acid sequence of BOP enables the construction of pools of nucleic acid probes encoding peptide fragments. Also, a consensus nucleic acid sequence designed as disclosed herein based on the amino acid sequence data, inferred in codons for the sequences, and observation of partial homology with known genes, also has been used as a probe. The probes may be used to isolate naturally occuring cDNAs which encode active mammalian osteogenic proteins (OP) as described below using standard hybridization methodology. The mRNAs are present in the cytoplasm of cells of various species which are known to synthesize osteogenic proteins.
Useful cells harboring the mRNAs include, for example, osteoblasts from bone or osteosarcoma, hypertrophic chondrocytes, and stem cells. The mRNAs can be used to produce cDNA libraries.
Alternatively, relevant DNAs encoding osteogenic protein may be retrieved from cloned genomic DNA
libraries from various mammalian species.
These discoveries enable the construction of DNAs encoding totally novel, non-native protein constructs which individually, and combined are capable of producing true endochondral bone. They also permit expression of the natural material, truncated forms, muteins, analogs, fusion proteins, 1 and various other variants and constructs, from cDNAs retrieved from natural sources or synthesized using the techniques disclosed herein using automated, commercially available equipment. The DNAs may be expressed using well established recombinant DNA
technologies in procaryotic or eucaryotic host cells, and may be oxidized and refolded in vitro if necessary for biological activity.

The isolation procedure for obtaining the protein from bone, the retrieval of an osteogenic protein gene, the design and production of recombinant protein, the nature of the matrix, and other material aspects concerning the nature, utility, how to make, and how to use the subject matter claimed herein will be further understood from the following, which constitutes the best method currently known for practicing the various aspects of the invention.

Al. Preparation of Demineralized Bone Demineralized bovine bone matrix is prepared by previously published procedures (Sampath and Reddi (1983) Proc. Natl. Acad. Sci. USA jQ:6591-6595).
Bovine.diaphyseal bones (age 1-10 days) are obtained from a local slaughterhouse and used fresh. The bones are stripped of muscle and fat, cleaned of periosteum, demarrowed by pressure with cold water, dipped in cold absolute ethanol, and stored at -20 C. They are then dried and fragmented by crushing and pulverized in a large mill. Care is taken to prevent heating by using liquid nitrogen.
The pulverized bone is milled to a particle size between 70-420 dun and is defatted by two washes of approximately two hours duration with three volumes of chloroform and methanol (3:1). The particulate bone is then washed with one volume of absolute ethanol and dried over one volume of anhydrous ether. The defatted bone powder (the alternative method is to obtain Bovine Cortical Bone Powder (75-425pm) from American Biomaterials) is then demineralized with 10 volumes of 0.5 N HC1 at 4 C for 40 min., four times. Finally, neutralizing washes are done on the demineralized bone powder with a large volume of water.

i t 1 A2. Dissociative Extraction and Ethanol Precipitation Demineralized bone matrix thus prepared is dissociatively extracted with 5 volumes of 4 M
guanidine-HC1, 50mM Tris-HC1, pH 7.0, containing protease inhibitors (5mM benzamidine, 44mM
6-aminohexanoic acid, 4.3mM N-ethylmaleimide, 0.44mM
phenylmethylsulfonyfluoride) for 16 hr. at 4 C. The suspension is filtered. The supernatant is collected and concentrated to one volume using an ultrafiltration hollow fiber membrane (Amicon, YM-10). The concentrate is centrifuged (8,000 x g for 10 min. at 4 C), and the supernatant is then subjected to ethanol precipitation. To one volume of concentrate is added five volumes of cold (-70 C) absolute ethanol (100%), which is then kept at -70 C
for 16 hrs. The precipitate is obtained upon centrifugation at 10,000 x g for 10 min. at 4 C. The resulting pellet is resuspended in 4 1 of 85% cold ethanol incubated for 60 min. at -700C and recentrifuged. The precipitate is again resuspended in 85% cold ethanol (2 1), incubated at -700C for 60 min. and centrifuged. The precipitate is then lyophilized.

A3. He arin-Sepharose Chromatography -I

The ethanol precipitated, lyophilized, extracted crude protein is dissolved in 25 volumes of 6 M urea, 50mM Tris-HC1, pH 7.0 (Buffer A) containing 0.15 M NaCl, and clarified by centrifugation at 8,000 x g for 10 min. The heparin-Sepharose*is Trade Mark C

1 column-equilibrated with Buffer A. The protein is loaded onto the column and after washing with three column volume of initial buffer (Buffer A containing 0.15 M NaCl), protein is eluted with Buffer A
containing 0.5 M NaCl. The absorption of the eluate is monitored continuously at 280 nm. The pool of protein eluted by 0.5 M NaCl (approximately 1 column volumes) is collected and stored at 4 C.

As shown in FIGURE 2A, most of the protein (about 95%) remains unbound. Approximately 5% of the protein is bound to the column. The unbound fraction has no bone inductive activity when bioassayed as a whole or after a partial purification through Sepharose CL-6B.

A4. Hydro Chromatography The volume of protein eluted by Buffer A
containing 0.5 M NaCl from the heparin-Sepharose*is applied directly to a column of hydroxyapaptite-ultrogel (HAP-ultrogel) (LKB Instruments), equilibrated with Buffer A containing 0.5 M NaCl.
The HAP-ultrogel is treated with Buffer A containing 500mM Na phosphate prior to equilibration. The unadsorbed protein is collected as an unbound fraction, and the column is washed with three column volumes of Buffer A containing 0.5 M NaCl. The column is subsequently eluted with Buffer A
containing 100mM Na Phosphate (FIGURE 2B).
Trade Mark - -1 The eluted component can induce endochondral bone as measured by alkaline phosphatase activity and histology. As the biologically active protein is bound to HAP in the presence of 6 M urea and 0.5 M
NaCl, it is likely that the protein has an affinity for bone mineral and may be displaced only by phosphate ions.

A5. Sephacryl*S-300 Gel Exclusion Chromatography Sephacryl*5-300 HR (High Resolution, 5 cm x 100 cm column) is obtained from Pharmacia and equilibrated with 4 M guanidine-HC1, 50mM Tris-HC1, pH 7Ø The bound protein fraction from HA-ultrogel is concentrated and exhanged from urea to 4 M
guanidine-HC1, 50mM Tris-HC1, pH 7.0 via an Amicon ultrafiltration YM-10 membrane. The solution is then filtered with Schleicher and Schuell CENTREX*
disposable microfilters. A sample aliquot of approximately 15 ml containing approximately 400 mg of protein is loaded onto the column and then eluted with 4 M guanidine-HC1, 50mM Tris-HC1, pH 7.0, with a flow rate of 3 ml/min; 12 ml fractions are collected over 8 hours and the concentration of protein is measured at A280nm (FIGURE 2C). An aliquot of the individual fractions is bioassayed for bone formation. Those fractions which have shown bone formation and have a molecular weigh less than 35kD
are pooled and concentrated via an Amicon*
ultrafiltration system with YM-10 membrane.
* Trade-mark 1 A6. Heparin-Sepharose Chromatography-II

The pooled osteo-inductive fractions obtained from gel exclusion chromatography are dialysed extensively against distilled water and then against 6 M urea, 50mM Tris-HC1, pH 7.0 (Buffer A) containing 0.1 M NaCl. The dialysate is then cleared through centrifugation. The sample is applied to the heparin-sepharose column (equilibrated with the same buffer). After washing with three column volumes of initial buffer, the column is developed sequentially with Buffer B containing 0.15 M NaCl, and 0.5 M NaCl (FIGURE 2D). The protein eluted by 0.5 M NaCl is collected and dialyzed extensively against distilled water. it is then dialyzed against 30% acetonitrile, 0.1% TFA at 40C.

A7. Reverse Phase HPLC

The protein is further purified by C-18 Vydac*silica-based HPLC column chromatography (particle size 5 }&m; pore size 300 A). The osteoinductive fraction obtained from heparin-sepharose-II chromatograph is loaded onto the column, and washed in 0.1% TFA, 10% acetonitrile for five min. As shown in FIGURE 8, the bound proteins are eluted with a linear gradient of 10-30%
acetonitrile over 15 min., 30-50% acetonitrile over 60 min, and 50-70% acetonitrile over 10 min at 22 C
with a flow rate of 1.5 ml/min and 1.4 ml samples are collected in polycarbonate tubes. Protein is * Trade-mark I

1 monitored by absorbance at A214 nm. Column fractions are tested for the presence of osteoinductive activity, concanavalin A-blottable proteins and then pooled. Pools are then characterized biochemically for the presence of 30 kD
protein by autoradiography, concanavalin A blotting, and Coomassie blue dye staining. They are then assayed for in vivo osteogenic activity. Biological activity is not found in the absence of 30 kD protein.
A8. Gel Elution The glycosylated or deglycosylated protein is eluted from SDS gels (0.5 mm and 1.5 mm thickness) for further characterization. 1251-labelled 30 kD
protein is routinely added to each preparation to monitor yields. TABLE 1 shows the various elution buffers that have been tested and the yields of 125I-labelled protein.

Elution of 30 kD Protein from SDS Gel % Eluted Buffer 0.5mm 1mm (1) dH2O 22 (2) 4M Guanidine-HC1, Tris-HC1, pH 7.0 2 (3) 4M Guanidine-HC1, Tris-HC1, pH 7.0, 93 52 0.5% Triton x 100 (4) 0.1% SDS, Tris-HC1, pH 7.0 98 1 TABLE 2 lists the steps used to isolate the 30 kD or deglycosylated 27 kD gel-bound protein. The standard protocol uses diffusion elution using 4M
*
guanidine-HC1 containing 0.5% Triton x 100 in Tris-HC1 buffer or in Tris-HC1 buffer containing 0.1%
SDS to achieve greater than 95% elution of the protein from the 27 or 30 kD region of the gel for demonstration of osteogenic activity in vivo as described in later section.

Preparation of Gel Eluted Protein C-18 Pool or deglycoslated protein plus '25I-labelled 30 kD protein) 1. Dry using vacuum centrifugation;
2. Wash pellet with H20;
3. Dissolve pellet in gel sample buffer (no reducing agent);
4. Electrophorese on pre-electrophoresed 0.5 mm mini gel;
..5. Cut out 27 or 30 kD protein;
6. Elute from gel with 0.1% SDS, 50mM Tris-HC1, pH
7.0;
7. Filter through Centrex*membrane;
8. Concentrate in Centricon tube (10 kD membrane);
9. Chromatograph of TSK-3000 gel filtration column;
10. Concentrate in Centricon*tube.

* Trade-mark 1 Chromatography in 0.1% SDS on a TSK-3000 gel filtration column is performed to separate gel impurities, such as soluble acrylamide, from the final product. The overall yield of labelled 30 kD
protein from the gel elution protocol is 50 - 60% of the loaded sample. Most of the loss occurs in the electrophoresis step, due to protein aggregation and/or smearing.

The yield is 0.5 to 1.0 pg substantially pure osteogenic protein per kg of bone.

A9. Isolation of the 16 kD and 18 kD Species TABLE 3 summarizes the procedures involved in the preparation of the subunits. Approximately 10 Kg of gel eluted 30 kD protein (FIGURE 3) is carboxymethylated and electrophoresed on an SDS-gel.
The sample contains 125I-label to trace yields and to use as an indicator for slicing the 16 kD, 18 kD
and non-reduceable 30 K regions from the gel. FIGURE
15 shows a Coomassie stained gel of aliquots of the protein isolated from the different gel slices. The slices corresponding to the 16 kD, 18 kD and non-reduceable 30 kD species contained approximately 2-3 pg, 3-4 pg, and 1-2 Ng, of protein respectively, as estimated by staining intensity.
Prior to SDS electrophoresis, all of the 30 kD
species can be reduced to the 16 kD and 18 kD
species. The nonreducible 30 kD species observed after electrophoresis appears to be an artifact resulting from the electrophoresis procedure.

Isolation of the Subunits of the 30 kD protein (C-18 pool plus 1251 labeled 30 kD protein) 1. Electrophorese on SDS gel.
2. Cut out 30 kD protein.
3. Elute with 0.1% SDS, 50 nm Tris, pH 7Ø
4. Concentrate and wash with H 20 in Centricon tube (10 kD membranes).
5. Reduce and carboxymethylate in 1% SDS, 0.4 M
Tris, pH 8.5.
6. Concentrate and wash with H 20 in Centricon tube.
7. Electrophorese on SDS gel.
8. Cut out the 16 kD and 18 kD subunits.
9. Elute with 0.1% SDS, 50mM Tris, pH 7Ø
10. Concentrate and wash with H2O in Centricon tubes.

B. Biological Characterization of BOP
B1. Gel Slicing:

Gel slicing experiments confirm that the isolated 30 kD protein is the protein responsible for osteogenic activity.

purification 1 Gels from the last step of the are sliced. Protein in each fraction is extracted in 15mM Tris-HC1, pH 7.0 containing 0.1% SDS or in buffer containing 4M guanidine-HC1, 0.5% non-ionic detergent (Triton x 100), 50 mM Tris-HC1. The extracted proteins are desalted, concentrated, and assayed for endochondral bone formation activity.
The results are set forth in FIGURE 14. From this Figure it is clear that the majority of osteogenic activity is due to protein at 30kD region of the gene. Activity in higher molecular weight regions is apparently due to protein aggregation. These protein aggregates, when reduced, yields the 16 kD and 18 kD
species discussed above.
*
B2. Con A-Sepharose Chromatography:

A sample containing the 30 kD protein is solubilized using 0.1% SDS, 50mM Tris-HC1, and is applied to a column of Con A-Sepharose*equilibrated with the same buffer. The bound material is eluted in SDS Tris-HC1 buffer containing 0.5 M alpha-methyl mannoside. After reverse phase chromatography of both the bound and unbound fractions, Con A-bound materials, when implanted, result in extensive bone formation. Further characterization of the bound materials show a Con A-blottable 30 kD protein.
Accordingly, the 30 kD glycosylated protein is responsible for the bone forming activity.
* Trade-mark r V

r Y

1 B3. Gel Permeation Chromatography:

TSK-3000/2000 gel permeation chromatography in guanidine-HC1 alternately is used to achieve separation of the high specific activity fraction *
obtained from C-18'chromatography (FIGURE 9). The results demonstrate that the peak of bone inducing activity elutes in fractions containing substantially pure 30 kD protein by Coomassie blue staining. When this fraction is iodinated and subjected to autoradiography, a strong band at 30 kD accounts for 90% of the iodinated proteins. The fraction induces bone formation in vivo at a dose of 50 to 100 ng per implant.
B4. Structural Requirements for Biological Activity B4-1 Activity after Digestion Although the role of 30 kD osteogenic protein is clearly established for bone induction, through analysis of proteolytic cleavage products we have begun to search for a minimum structure that is necessary for activity j viva. The results of cleavage experiments demonstrate that pepsin treatment fails to destroy bone inducing capacity, whereas trypsin or CNBr completely abolishes the activity.

* Trade-mark 1 An experiment is performed to isolate and identify pepsin digested product responsible for biological activity. Sample used for pepsin digest were 20% - 30% pure. The buffer used is 0.1% TFA in water. The enzyme to substrate ratio is 1:10. A
control sample is7made without enzyme. The digestion mixture is incubated at room temperature for 16 hr.
The digested product is then separated in 4 M
guanidine-HC1 using gel permeation chromatography, and the fractions are prepared for in vivo assay.
The results demonstrate that active fractions from gel permeation chromotography of the pepsin digest correspond to molecular weight of 8 kD - 10 kD.

B4-2 Unalycosylated Protein is Active In order to understand the importance of the carbohydrates moiety with respect to osteogenic activity, the 30 kD protein has been chemically deglycosylated using HF (see below). After analyzing an aliquot of the reaction product by Con A blot to confirm the absence of carbohydrate, the material is assayed for its activity in vivo. The bioassay is positive (i.e., the deglycosylated protein produces a bone formation response as determined by histological examination shown in FIGURE 17C), demonstrating that exposure to HF did not destroy the biological function of the protein, and thus that the OP does not require carboyhdrate for biological activity. In addition, the specific activity of the deglycosylated protein is approximately the same as that of the native glycosylated protein.

1 B5. Specific Activity of BOP

Experiments were performed 1) to determine the half maximal bone-inducing activity based on calcium content of the implant; 2) to estimate proteins at nanogram levels using a gel scanning method; and 3) to establish dose for half maximal bone inducing activity for gel eluted 30kD BOP. The results demonstrate that gel eluted substantially pure 30kD osteogenic protein induces bone at less than 5 ng per 25 mg implant and exhibits half maximal bone differentiation activity at 20 ng per implant.
The purification data suggest that osteogenic protein has been purified from bovine bone to 367,307 fold after final gel elution step with a specific activity of 47,750 bone forming units per mg of protein.
B5(a)Half Maximal Bone Differentiation Activity The bone inducing activity is determined biochemically by the specific activity of alkaline phosphatase and calcium content of the day 12 implant. An increase in the specific activity of alkaline phosphatase indicates the onset of bone formation. Calcium content, on the other hand, is proportional to the amount of bone formed in the implant. The bone formation is therefore calculated by determining calcium content of the implant on day 12 in rats and expressed as bone forming units, which represent the amount that exhibits half maximal bone inducing activity compared to rat demineralized bone matrix. Bone induction - -1 exhibited by intact demineralized rat bone matrix is considered to be the maximal bone-differentiation activity for comparison.

B5(b)Protein Estimation Using Gel Scanning Techniques A standard curve is developed employing known amounts of a standard protein, bovine serum albumin. The protein at varying concentration (50-300 ng) is loaded on 15% SDS gel, electrophoresed, stained in comassie and destained.
The gel containing standard proteins is scanned at predetermined settings using a gel scanner at 580 nm. The area covered by the protein band is calculated and a standard curve against concentrations of protein is constructed. A sample with an unknown protein concentration is electrophoresed with known concentration of BSA. The lane contained unknown sample is scanned and from the area the concentration of protein is determined.
B5(c)Gel Elution and Specific Activity An aliquot of C-18 highly purified active fraction is subjected to SDS gel and sliced according to molecular weights described in Figure 14.
Proteins are eluted from the slices in 4 M
guanidine-HC1 containing 0.5% Triton X-100, desalted, concentrated and assayed for endochondral bone forming activity as determined by calcium content.
The C-18 highly active fractions and gel eluted Trade Mark 1 substantially pure 30 kD osteogenic protein are implanted in varying concentrations in order to determine the half maximal bone inducing activity.

Figure 14 shows that the bone inducing activity is due to proteins eluted in the 28-34 kD
region. The recovery of activity after gel elution step is determined by calcium content. Figures 19A
and 19B represent the bone inducing activity for the various concentrations of 30 kD protein before and after gel elution as estimated by calcium content.
The data suggest that the half maximal activity for 30 kD protein before gel elution is 69 nanogram per 25 mg implant and is 21 nanogram per 25 mg implant after elution. Table 4 describes the yield, total specific activity, and fold purification of osteogenic protein at each step during purification.
Approximately 500 ug of heparin sepharose I fraction, 130-150 ug of the HA ultrogel fraction, 10-12 ug of the gel filtration fraction, 4-5 ug of the heparin sepharose II fraction, 0.4-0.5 ug of the C-18 highly purified fraction, and 20-25 ng of the gel eluted, substantially purified fraction is needed per 25 mg of implant for unequivocal bone formation for half maximal activity. Thus, 0.8-1.0 ng purified osteogenic protein per mg. of implant is required to exhibit half maximal bone differentiation activity in vivo.

PURIFICATION OF BOP

Purification Protein Biological Specific Purification Steps (mg.) Activity Activity Fold Units* Units/mg.
Ethanol Precipitate** 30,000# 4,000 0.13 1 Heparin Sepharose*I 1,200# 2,400 2.00 15 HA-Ultrogel 300# 2,307 7.69 59 Gel filtration 20# 1,600 80.00 615 Heparin Sepharose*II 5# 1,000 200.00 1,538 C-18 HPLC 0.070@ 150 2,043.00 15,715 Gel elution 0.004@ 191 47,750.00 367,307 Values are calculated from 4 kg. of bovine bone matrix (800 g of demineralized matrix).

* One unit of bone forming activity is defined as the amount that exhibits half maximal bone differentiation activity compared to rat demineralized bone matrix, as determined by calcium content of the implant on day 12 in rats.

# Proteins were measured by absorbance at 280 nm.
*
Trade Mark 1 @ Proteins were measured by gel scanning method compared to known standard protein, bovine serum albumin.

** Ethanol-precipitated guanidine extract of bovine bone is a weak inducer of bone in rats, possibly due to endogenous inhibitors. This precipitate is subjected to gel filtration and proteins less than 50 kD were separated and used for bioassay.

C. CHEMICAL CHARACTERIZATION OF BOP
Cl. Molecular Weight and Structure Electrophoresis of the most active fractions from reverse phase C-18 chromatography on non-reducing SDS polyacrylamide gels reveals a single band at about 30 kD as detected by both Coomassie blue staining (FIGURE 3A) and autoradiography.

In order to extend the analysis of BOP, the protein was examined under reducing conditions.
FIGURE 3B shows an SDS gel of BOP in the presence of dithiothreitol. Upon reduction, 30 kD BOP yields two species which are stained with Coomassic blue dye: a 16 kD species and an 18 kD species. Reduction causes loss of biological activity. The two reduced BOP
species have been analyzed to determine if they are structurally related. Comparison of the amino acid composition of the two proteins (as disclosed below) shows little differences, indicating that the native protein may comprise two chains having some homology.
C2. Charge Determination Isoelectric focusing studies are initiated to further evaluate the 30 kD protein for possibile heterogeneity. Results to date have not revealed any such heterogeneity. The oxidized and reduced species migrate as diffuse bands in the basic region of the 1 isoelectric focusing gel, using the iodinated 30 kD
protein for detection. Further analysis using two dimensional gel electrophoresis and Con A for detection indicate that the oxidized 30 kD protein migrates as one species in the same basic region as the iodinated 30 kD protein. The diffuse character of the band may be traced to the presence of carbohydrate attached to the protein.

C3. Presence of Carbohydrate The 30 kD protein has been tested for the presence of carbohydrate by Concanavalin A (Con A) blotting after SDS-PAGE and transfer to nitrocellulose paper. The results demonstrate that the 30 kD protein has a high affinity for Con A, indicating that the protein is glycosylated (FIGURE
4A). In addition, the Con A blots provide evidence for a substructure in the 30 kD region of the gel, suggesting heterogeneity due to varying degrees of glycosylation. After reduction (FIGURE 4B), Con A
blots show evidence for two major components at 16 kD
and 18 kD. In addition, it has been demonstrated that no glycosylated material remains at the 30 kD
region after reduction.

In order to confirm the presence of carbohydrate and to estimate the amount of carbohydrate attached, the 30 kD protein is treated with N-glycanase, a deglycosylating enzyme with a broad specificity. Samples of the 125I-labelled 30 kD protein are incubated with the enzyme in the presence of SDS for 24 hours at 37 C. As observed by SDS-PAGE, the treated samples appear as a prominent species at about 27 kD (FIGURE 5A). Upon reduction, the 27 kD species is reduced to species having a molecular weight of about 14 kD - 16 kD (FIGURE 5B).

To ensure complete deglycosylation of the 30KD protein, chemical cleavage of the carbohydrate moieties using hydrogen fluoride (HF) is performed.
Active osteogenic protein fractions pooled from the C-18 chromatography step are dried in vacuo over P205 in a polypropylene tube, and 50 }il freshly distilled anhydrous HF at -70 C is added. After capping the tube tightly, the mixture is kept at 0 C
in an ice-bath with occasional agitation for 1 hr.
The HF is then evaporated using a continuous stream of dry nitrogen gas. The tube is removed from the ice bath and the residue dried in vacuo over P205 and KOH pellets.

Following drying, the samples are dissolved in 100 p1 of 50% acetonitrile/0.1% TFA and aliquoted for SDS gel analysis, Con A binding, and biological assay. Aliquots are dried and dissolved in either SDS gel sample buffer in preparation for SDS gel analysis and Con A blotting or 4 M
guanidine-HC1, 50mM Tris-HC1, pH 7.0 for biological assay.

The results show that samples are completely deglycosylated by the HF treatment: Con A blots after SDS gel electrophoreses and transfer to Immobilon*

* Trade-mark 1 membrane showed no binding of Con A to the treated samples, while untreated controls were strongly positive at 30 kD. Coomassie gels of treated samples showed the presense of a 27 kD band instead of the 30 kD band present in the untreated controls.
C4. Chemical and Enzymatic Cleavage Cleavage reactions with CNBr are analyzed using Con A binding for detection of fragments associated with carbohydrate. Cleavage reactions are conducted using trifluoroacetic acid (TFA) in the presence and absence of CNBr. Reactions are conducted at 37 C for 18 hours, and the samples are vacuum dried. The samples are washed with water, dissolved in SDS gel sample buffer with reducing agent, boiled and applied to an SDS gel. After electrophoresis, the protein is transferred to Immobilon membrane and visualized by Con A binding.
In low concentrations of acid (1%), CNBr cleaves the majority of 16 kD and 18 kD species to one product, a species about 14 kD. In reactions using 10% TFA, a 14 kD species is observed both with and without CNBr.

Four proteolytic enzymes are used in these experiments to examine the digestion products of the kD protein: 1) V-8 protease; 2) Endo Lys C
protease; 3) pepsin; and 4) trypsin. Except for pepsin, the digestion buffer for the enzymes is 0.1 M
30 ammonium bicarbonate, pH 8.3. The pepsin reactions are done in 0.1% TFA. The digestion volume is 100 p1 and the ratio of enzyme to substrate is 1:10.

1 125I-labelled 30 kD osteogenic protein is added for detection. After incubation at 37 C for 16 hr., digestion mixtures are dried down and taken up in gel sample buffer containing dithiothreitol for SDS-PAGE. FIGURE 6 shows an autoradiograph of an SDS
gel of the digestion products. The results show that under these conditions, only trypsin digests the reduced 16 kD/18 kD species completely and yields a major species at around 12 kD. Pepsin digestion yields better defined, lower molecular weight species. However, the 16 kD/18 kD fragments were not digested completely. The V-8 digest shows limited digestion with one dominant species at 16 kD.

C5. Protein Sequencing To obtain amino acid sequence data, the protein is cleaved with trypsin or Endoproteinase Asp-N (EndoAsp-N). The tryptic digest of reduced and carboxymethylated 30 kD protein (approximately 10 pg) is fractionated by reverse-phase HPLC using a C-8 narrowbore column (13 cm x 2.1 mm ID) with a TFA/acetonitrile gradient and a flow rate of 150 pl/min. The gradient employs (A) 0.06% TFA in water and (B) 0.04% TFA in water and acetonitrile (1:4; v:v). The procedure was 10% B for five min., followed by a linear gradient for 70 min. to 80% B, followed by a linear gradient for 10 min. to 100% B.
Fractions containing fragments as determined from the peaks in the HPLC profile (FIGURE 7A) are rechromatographed at least once under the same conditions in order to isolate single components satisfactory for sequence analysis.

1 The HPLC profiles of the similarly digested 16 kD and 18 kD subunits are shown in FIGUREs 7B and 7C, respectively. These peptide maps are similar suggesting that the subunits are identical or are closely related.

The 16 kD and 18 kD subunits are digested with Endo Asp N proteinase. The protein is treated with 0.5 pg EndoAsp-N in 50mM sodium phosphate buffer, pH 7.8 at 36 C for 20 hr. The conditions for fractionation are the same as those described previously for the 30 kD, 16 kD, and 18 kD digests.
The profiles obtained are shown in FIGUREs 16A and 16B.
Various peptide fragments produced using the foregoing procedures have been analyzed in an automated amino acid sequencer (Applied Biosystems 470A with 120A on-line PTH analysis). The following sequence data has been obtained:

(1) S-F-D-A-Y-Y-C-S-G-A-C-Q-F-P-M-P-K;
(2) S-L-K-P-S-N-Y-A-T-I-Q-S-I-V;
(3) A-C-C-V-P-T-E-L-S-A-I-S-M-L-Y-L-D-E-N-E-K;
(4) M-S-S-L-S-I-L-F-F-D-E-N-K;
(5) S-Q-E-L-Y-V-D-F-Q-R;
(6) F-L-H-C-Q-F-S-E-R-N-S;
(7) T-V-G-Q-L-N-E-Q-S-S-E-P-N-I-Y;
(8) L-Y-D-P-M-V-V;
(9) V-G-V-V-P-G-I-P-E-P-C-C-V-P-E;
(10) V-D-F-A-D-I-G;

1 (11) V-P-K-P-C-C-A-P-T;
(12) I-N-I-A-N-Y-L;
(13) D-N-H-V-L-T-M-F-P-I-A-I-N;
(14) D-E-Q-T-L-K-K-A-R-R-K-Q-W-I-?-P;
(15) D-I-G-?-S-E-W-I-I-?-P;
(16) S-I-V-R-A-V-G-V-P-G-I-P-E-P-?-?-V;
(17) D-?-I-V-A-P-P-Q-Y-H-A-F-Y;
(18) D-E-N-K-N-V-V-L-K-V-Y-P-N-M-T-V-E;
(19) S-Q-T-L-Q-F-D-E-Q-T-L-K-?-A-R-?-K-Q;
(20) D-E-Q-T-L-K-K-A-R-R-K-Q-W-I-E-P-R-N-?-A-R-R-Y-L;
(21) A-R-R-K-Q-W-I-E-P-R-N-?-A-?-R-Y-?-?-V-D; and (22) R-?-Q-W-I-E-P-?-N-?-A-?-?-Y-L-K-V-D-?-A-?-?-G.
C6. Amino Acid Analysis Samples of oxidized (30 kD) and reduced (16 kD and 18 kD) BOP are electrophoresed on a gel and transferred to Immobilon for hydrolysis and amino acid analysis using conventional, commercially available reagents to derivatize samples and HPLC
using the PicO Tag (Millipore) system. The composition data generated by amino acid analyses of 30 kD BOP is reproducible, with some variation in the number of residues for a few amino acids, especially cysteine and isoleucine.

* Trade-mark 1 Composition data obtained are shown in TABLE
5.

BOP Amino Acid Analyses Amino Acid 30 kD 16 kD 18 kD
Aspartic Acid/ 22 14 15 Asparagine Glutamic Acid/ 24 14 16 Glutamine Serine 24 16 23 Glycine 29 18 26 Histidine 5 * 4 Arginine 13 6 6 Threonine 11 6 7 Alanine 18 11 12 Proline 14 6 6 Tyrosine 11 3 3 Valine. 14 8 7 Methionine 3 0 2 Cysteine** 16 14 12 Isoleucine 15 14 10 Leucine 15 8 9 Phenylalanine 7 4 4 Tryptophan ND ND ND
Lysine 12 6 6 1 *This result is not integrated because histidine is present in low quantities.
**Cysteine is corrected by percent normally recovered from performic acid hydrolysis of the standard protein.

The results obtained from the 16 kD and 18 kD subunits, when combined, closely resemble the numbers obtained from the native 30 kD protein. The high figures obtained for glycine and serine are most likely the result of gel elution.

D. PURIFICATION OF HUMAN OSTEOGENIC PROTEIN
Human bone is obtained from the Bone Bank, (Massachusetts General Hospital, Boston, MA), and is milled, defatted, demarrowed and demineralized by the procedure disclosed above. 320 g of mineralized bone matrix yields 70 - 80 g of demineralized bone matrix. Dissociative extraction and ethanol precipitation of the matrix gives 12.5 g of guanidine-HC1 extract.

One third of the ethanol precipitate (0.5 g) is used for gel filtration through 4 M guanidine-HC1 (FIGURE 10A). Approximately 70-80 g of ethanol precipitate per run is used. In vivo bone inducing activity is localized in the fractions containing proteins in the 30 kD range. They are pooled and equilibrated in 6 M urea, 0.5 M NaCl buffer, and applied directly onto a HAP column; the bound protein 1 is eluted stepwise by using the same buffer containing 100mM and 500mM phosphate (FIGURE lOB). Bioassay of HAP
bound and unbound fractions demonstrates that only the fraction eluted by 100mM phosphate has bone inducing activity in vivo. The biologically active fraction obtained from HAP chromatography is subjected to heparin-Sepharose affinity chromatography in buffer containing low salt; the bound proteins are eluted by 0.5 M NaCl (Figure 10D. Figure 10C describes the elution profile for the intervening gel filtration step described on pp. 20 and 29, supra.). Assaying the heparin-Sepharose fractions shows that the bound fraction eluted by 0.5 M NaCl have bone-inducing activity. The active fraction is then subjected to C-18 reverse phase chromatography.
The active fraction can then be subjected to SDS-PAGE as noted above to yield a band at about 30 kD comprising substantially pure human osteogenic protein.
E. BIOSYNTHETIC PROBES FOR ISOLATION OF GENES
ENCODING NATIVE OSTEOGENIC PROTEIN

A synthetic consensus gene shown in FIGURE
13 was designed as a hybridization probe based on amino acid predictions from homology with the TGF-beta gene family and using human codon bias as found in human TGF-beta. The designed concensus sequence was then constructed using known techniques involving assembly of oligonucleotides manufactured in a DNA synthesizer.

1 Tryptic peptides derived from BOP and sequenced by Edman degradation provided amino acid sequences that showed strong homology with the Drosophila DPP protein sequence (as inferred from the gene), the Kenopus VG1 protein, and somewhat.less homology to inhibin and TGF-beta, as demonstrated below in TABLE 6.

protein amino acid sequence homology (BOP) SFDAYYCSGACQFPS
***** * * ** (9/15 matches) (DPP) GYDAYYCHGKCPFFL

(BOP) SFDAYYCSGACQFPS
* ** * * * (6/15 matches) (Val) GYMANYCYGECPYPL

(BOP) SFDAYYCSGACQFPS
* ** * * (5/15 matches) (inhibin) GYHANYCEGECPSHI

(BOP) SFDAYYCSGACQFPS
* * * * (4/15 matches) (TGF-beta) GYHANFCLGPCPYIW

(BOP) K/RACCVPTELSAISMLYLDEN
***** * **** * * (12/20 matches) (Val) LPCCVPTKMSPISMLFYDNN

1 (BOP) K/RACCVPTELSAISMLYLDEN
* ***** * **** * (12/20 matches) (inhibin) KSCCVPTKLRPMSMLYYDDG

(BOP) K/RACCVPTELSAISMLYLDE
**** * * (6/19 matches) (TGF-beta) APCCVPQALEPLPIVYYVG

(BOP) K/RACCVPTELSAISMLYLDEN
******* * **** (12/20 matches) (DPP) KACCVPTQLDSVAMLYLNDQ

(BOP) LYVDF
***** (5/5 matches) (DPP) LYVDF

(BOP) LYVDF
*** * (4/5 matches) (Val) LYVEF

(BOP) LYVDF
** ** (4/5 matches) (TGF-beta) LYIDF

( Q ) LYVDF
* * (2/4 matches) (inhibin) FFVSF

*-match 1 In determining the amino acid sequence of an osteogenic protein (from which the nucleic acid sequence can be determined), the following points were considered: (1) the amino acid sequence determined by Edman degradation of osteogenic protein tryptic fragments is ranked highest as long as it has a strong signal and shows homology or conservative changes when aligned with the other members of the gene family; (2) where the sequence matches for all four proteins, it is used in the synthetic gene sequence; (3) matching amino acids in DPP and Vgl are used; (4) If Vgl or DPP diverged but either one were matched by inhibin or by TGF-beta, this matched amino acid is chosen; (5) where all sequences diverged, the DPP sequence is initially chosen, with a later plan of creating the Vgl sequence by mutagenesis kept as a possibility. In addition, the consensus sequence is designed to preserve the disulfide crosslinking and the apparent structural homology.
One purpose of the originally designed synthetic consensus gene sequence, designated COPO, (see Fig. 13), was to serve as a probe to isolate natural genes. For this reason the DNA was designed using human codon bias. Alternatively, probes may be constructed using conventional techniques comprising a group of sequences of nucleotides which encode any portion of the amino acid sequence of the osteogenic protein produced in accordance with the foregoing isolation procedure. Use of such pools of probes also will enable isolation of a DNA encoding the intact protein.

50 _ 13 4610 1 E-2 Retrieval of Genes Encoding Osteogenic Protein from Genomic Library A human genomic library (Maniatis-library) carried in lambda phage (Charon 4A) was screened using the COPO consensus gene as probe. The initial screening was of 500,000 plaques (10 plates of 50,000 each). Areas giving hybridization signal were punched out from the plates, phage particles were eluted and plated again at a density of 2000-3000 plaques per plate. A second hybridization yielded plaques which were plated once more, this time at a density of ca 100 plaques per plate allowing isolation of pure clones. The probe (COPO) is a 300 base pair BamHI-PstI fragment restricted from an amplification plasmid which was labeled using alpha 32 dCTP according to the random priming method of Feinberg and Vogelstein, Anal. Biochem., 137, 266-267, 1984. Prehybridization was done for 1 hr in 5x SSPE, lOx Denhardt's mix, .5% SDS at 50 C.
Hybridization was overnight in the same solution as above plus probe. The washing of nitrocellulose membranes was done, once cold for 5 min. in lx SSPE
with .1% SDS and twice at 500C for 2x30 min. in the same solution. Using this procedure, twenty-four positive clones were found. Two contained a gene never before reported designated OP1, osteogenic protein-i described below. Two others yielded the genes corresponding to BMP-2b, one yielded BMP-3 (see U.S. Patent No. 5,011,691, issued April 30, 1991).

1 Southern blot analysis of lambda #13 DNA
showed that an approximately 3kb BamHI fragment hybridized to the probe. (See Fig. 1B). This fragment was isolated and subcloned into a.Bluescript*
vector (at the BamHI site). The clone was further analyzed by Southern blotting and hybridization to the COPO probe. This showed that a lkb (approx.) EcoRI fragment strongly hybridized to the probe.
This fragment was subcloned into the EcoRI site of a Bluescript*vector, and sequenced. Analysis of this sequence showed that the fragment encoded the carboxy terminus of a protein, named osteogenic protein-1 (OP1). The protein was identified by amino acid homology with the TGF-beta family. For this comparison cysteine patterns were used and then the adjacent amino acids were compared. Consensus splice signals were found where amino acid homologies ended, designating exon intron boundaries. Three exons were combined to obtain a functional TGF-beta-like domain containing seven cysteines. Two introns were deleted by looping out via primers bridging the exons using the single stranded mutagenesis method of Kunkel.
Also, upstream of the first cysteine, an EcoRI site and an asp-pro junction for acid cleavage were introduced, and at the 3' end a PstI site was added by the same technique. Further sequence information (penultimate exon) was obtained by sequencing the entire insert. The sequencing was done by generating a set of unidirectionally deleted clones (Ozkaynak, E., and Putney, S.: Biotechniques, 5, 770-773, 1987). The obtained sequence covers about 80% of the TGF-beta-like region of OP1 and is set forth in FIG.
*
Trade Mark E

_52- 1341610 1 1A. The complete sequence of the TGF-beta like region was obtained by first subcloning all EcoRI
generated fragments of lambda clone #13 DNA and sequencing a 4kb fragment that includes the first portion of the TGF-beta like region (third exon counting from end) as well as sequences characterized earlier. The gene on an EcoRI to PstI fragment was inserted into an E. coli expression vector controlled by the trp promoter-operator to produce a modified trp LE fusion protein with an acid cleavage site.
The OP1 gene encodes amino acids corresponding substantially to a peptide found in sequences of naturally sourced material. The amino acid sequence of what is believed to be its active region is set forth below:

YMNATN--H-AIVQTLVHFINPET-VPKPCCAPTQLNA

ISVLYFDDSSNVILKKYRNMVVRACGCH

A longer active sequence is:

HQRQA

YMNATN--H-AIVQTLVHFINPET-VPKPCCAPTQLNA

ISVLYFDDSSNVILKKYRNMVVRACGCH

The amino acid sequence of what is believed to be the active regions encoded by the other three native genes retrieved using the consensus probe are:

CBMP-2a CKRHPLYVDFS-DVGWNDWIVAPPGYHAFYCHGECPFPLAD

HLNSTN--H-AIVQTLVNSVNS-K-IPKACCVPTELSA

ISMLYLDENEKVVLKNYQDMVVEGCGCR

CBMP-2b CRRHSLYVDFS-DVGWNDWIVAPPGYQAFYCHGDCPFPLAD

HLNSTN--H-AIVQTLVNSVNS-S-IPKACCVPTELSA

ISMLYLDEYDKVVLKNYQEMVVEGCGCR

SLKPSN--H-ATIQSIVRAVGVVPGIPEPCCVPEKMSS

LS ILFFDENKNVVLKVYPNMTVESCACR

E-3 Probing cDNA Library Another example of the use of pools of probes to enable isolation of a DNA encoding the intact protein is shown by the following. Cells known to express the protein are extracted to isolate total cytoplasmic RNA. An oligo-dT column can be used to isolate mRNA. This mRNA can be size fractionated by, for example, gel electrophoresis. The fraction which includes the mRNA of interest may be determined by inducing transient expression in a suitable host cell and testing for the presence of osteogenic protein using, for example, antibody raised against peptides derived from the tryptic fragments of osteogenic protein in an immunoassay. The mRNA
fraction is then reverse transcribed to single stranded cDNA using reverse transcriptase; a second complementary DNA strand can then be synthesized using the cDNA as a template. The double-standard DNA is then ligated into vectors which are used to transfect bacteria to produce a cDNA library.
The radiolabelled consensus sequence, portions thereof, and/or synthetic deoxy oligonucleotides complementary to codons for the known amino acid sequences in the osteogenic protein may be used to identify which of the DNAs in the cDNA library encode the full length osteogenic protein by standard DNA-DNA
hybridization techniques.

The cDNA may then be integrated in an expression vector and transfected into an appropriate host cell for protein expression. The host may be a prokaryotic or eucaryotic cell since the former's inability to glycosylate osteogenic protein will not effect the protein's enzymatic activity. Useful host cells include - -1 Saccharomyces, E, coli, and various mammalian cell cultures. The vector may additionally encode various signal sequences for protein secretion and/or may encode osteogenic protein as a fusion protein. After being translated, protein may be purified from the cells or recovered from the culture medium.

E4. Gene Preparation Natural gene sequences and cDNAs retrieved as described above may be used for expression. The genes above may also be produced by assembly of chemically synthesized oligonucleotides. 15-100mer oligonucleotides may be synthesized on a Biosearch DNA Model 8600 Synthesizer, and purified by polyacrylamide gel electrophoresis (PAGE) in Tris-Borate-EDTA buffer (TBE). The DNA is then electroeluted from the gel. Overlapping oligomers may be phosphorylated by T4 polynucleotide kinase and ligated into larger blocks which may also be purifed by PAGE.
E5. Expression The genes can be expressed in appropriate prokaryotic hosts such as various strains of Ei coli. For example, if the gene is to be expressed in E. coli, an expression vector based on pBR322 and containing a synthetic trp promoter operator and the modified trp LE leader maybe used. The vector can be opened at the EcoRI and PSTI

1 restriction sites, and, for example, an FB-FB-OP
gene fragment can be inserted between these sites, where FB is fragment B of Staphylococcal Protein A, and is used as a leader. The expressed fusion protein results from attachment of the OP gene to a fragment encoding FB. The OP protein is joined to the leader protein via a hinge region having the sequence asp-pro-asn-gly. This hinge permits chemical cleavage of the fusion protein with dilute acid at the asp-pro site or cleavage at asn-gly with hydroxylamine, resulting in release of the OP protein.

E6. Production of Active Proteins The following procedure may be followed for production of active recombinant proteins. E.
coli cells containing the fusion proteins are lysed. The fusion proteins are purified by differential solubilization. Cleavage is conducted with dilute acid, and the resulting cleavage products are passed through a Sephacryl-200HR or SP Trisacyl column to separate the cleaved proteins. The OP fractions are then subjected to HPLC on a semi-prep C-18 column. The HPLC column primarily separates the leader proteins and other minor impurities from the OP.

Initial conditions for refolding of OP
were at pH 8.0 using Tris, GuHC1, dithiothreitol.

1 Final conditions for refolding of OP analogs were at pH 8.0 using Tris, oxidized glutathione, and lower amounts of GuHC1 and dithiothreitol.

These procedures have been used to express in E coli on the active protein designated OP1 having the amino acid sequence set forth above (longer species).

A. General Consideration of Matrix Properties The carrier described in the bioassay section, infra, may be replaced by either a biodegradable-synthetic or synthetic-inorganic matrix (e.g., HAP, collagen, tricalcium phosphate, or polylactic acid, polyglycolic acid and various copolymers thereof). Also xenogeneic bone may be used if pretreated as described below.

Studies have shown that surface charge, particle size, the presence of mineral, and the methodology for combining matrix and osteogenic protein all play a role in achieving successful bone induction. Perturbation of the charge by chemical modification abolishes the inductive response.
Particle size influences the quantitative response of new bone; particles between 75 and 420 pm elicit the maximum response. Contamination of the matrix with bone mineral will inhibit bone formation. Most importantly, the procedures used to formulate osteogenic protein onto the matrix are extremely sensitive to the physical and chemical state of both the osteogenic protein and the matrix.

The sequential cellular reactions at the interface of the bone matrix/OP implants are complex. The multistep cascade includes: binding of fibrin and fibronectin to implanted matrix, - -chemotaxis of cells, proliferation of fibroblasts, differentiation into chondroblasts, cartilage formation, vascular invasion, bone formation, remodeling, and bone marrow differentiation.
A successful carrier for osteogenic protein must perform several important functions. It must bind osteogenic protein and act as a slow release delivery system, accommodate each step of the to cellular response during bone development, and protect the osteogenic protein from nonspecific proteolysis. In addition, selected materials must be biocompatible in vivo and biodegradable; the carrier must act as a temporary scaffold until replaced completely by new bone. Polylactic acid (PLA), polyglycolic acid (PGA), and various combinations have different dissolution rates in vivo. In bones, the dissolution rates can vary according to whether the implant is placed in cortical or trabecular bone.
Matrix geometry, particle size, the presence of surface charge, and porosity or the presence of interstices among the particles of a size sufficient to permit cell infiltration, are all important to successful matrix performance. It is preferred to shape the matrix to the desired form of the new bone and to have dimensions which span non-union defects.
Rat studies show that the new bone is formed essentially having the dimensions of the device implanted.

1 The matrix may comprise a shape-retaining solid made of loosely adhered particulate material, e.g., with collagen. It may also comprise a molded, porous solid, or simply an aggregation of close-packed particles held in place by surrounding tissue. Masticated muscle or other tissue may also be used. Large allogeneic bone implants can act as a carrier for the matrix if their marrow cavities are cleaned and packed with particles and the dispersed osteogenic protein.

B. Preparation of Biologically Active Aliovenic Matrix Demineralized bone matrix is prepared from the dehydrated diaphyseal shafts of rat femur and tibia as described herein to produce a bone particle size which pass through a 420 pm sieve. The bone particles are subjected to dissociative extraction with 4 M guanidine-HC1. Such treatment results in a complete loss of the inherent ability of the bone matrix to induce endochondral bone differentiation.
The remaining insoluble material is used to fabricate the matrix. The material is mostly collagenous in nature, and upon implantation, does not induce cartilage and bone. All new preparations are tested for mineral content and false positives before use.
The total loss of biological activity of bone matrix is restored when an active osteoinductive protein fraction or a pure protein is reconstituted with the a 1 - -biologically inactive insoluble collagenous matrix.
The osteoinductive protein can be obtained from any vertebrate, e.g., bovine, porcine, monkey, or human, or produced using recombinant DNA techniques.

C. Preparation of Deglycosylated Bone Matrix for Use in Xenogenic Implant When osteogenic protein is reconstituted with collagenous bone matrix from other species and implanted in rat, no bone is formed. This suggests that while the osteogenic protein is xenogenic (not species specific), the matrix is species specific and cannot be implanted cross species perhaps due to intrinsic immunogenic or inhibitory components.
Thus, heretofore, for bone-based matrices, in order for the osteogenic protein to exhibit its full bone inducing activity, a species specific collagenous bone matrix was required.
The major component of all bone matrices is Type I collagen, which is glycosylated. In addition to collagen, extracted bone includes non-collagenous proteins which may account for 5% of its mass. Many non-collagenous components of bone matrix are glycoproteins. Although the biological significance of the glycoproteins in bone formation is not known, they may present themselves as potent antigens by virtue of their carbohydrate content and may constitute immunogenic and/or inhibitory components that are present in xenogenic matrix.

- -1 It has now been discovered that a collagenous bone matrix may be used as a carrier to effect bone inducing activity in xenogenic implants, if one first removes the immonogenic and inhibitory components from the matrix. The matrix is deglycosglated chemically using, for example, hydrogen fluoride to achieve this purpose.

Bovine bone residue prepared as described above is sieved, and particles of the 74-420 pM are collected. The sample is dried vacuo over P205, transferred to the reaction vessel and anhydrous hydrogen fluoride (HF) (10-20 ml/g of matrix) is then distilled onto the sample at -70 C.
The vessel is allowed to warm to 00 and the reaction mixture is stirred at this temperature for 60 min.
After evaporation of the HF in vacuo, the residue is dried thoroughly in vacuo over KOH pellets to remove any remaining traces of acid.
Extent of deglycosylation can be determined from carbohydrate analysis of matrix samples taken before and after treatment with HF, after washing the samples appropriately to remove non-covalently bound carbohydrates.

The deglycosylated bone matrix is next treated as set forth below:

1) suspend in TBS (Tris-buffered Saline) lg/200 ml and stir at 4 C for 2 hrs;

1 2) centrifuge then treated again with TBS, lg/200 ml and stir at 4 C overnight; and 3) centrifuged; discard supernatant; water wash residue; and then lyophilized.

FABRICATION OF OSTEOGENIC DEVICE

Fabrication of osteogenic devices using any of the matrices set forth above with any of the osteogenic proteins described above may be performed as follows.

A. Ethanol Precipitation In this procedure, matrix was added to osteogenic protein in guanidine-HC1. Samples were vortexed and incubated at a low temperature. Samples were then further vortexed. Cold absolute ethanol was added to the mixture which was then stirred and incubated. After centrifugation (microfuge high speed) the supernatant was discarded. The reconstituted matrix was washed with cold concentrated ethanol in water and then lyophilized.
B. Acetonitrile Trifluoroacetic Acid Lvophilization In this procedure, osteogenic protein in an acetonitrile trifluroacetic acid (ACN/TFA) solution was added to the carrier. Sarpples were vigorously vortexed many times and then lyophilized.

_64- 1341610 1 C. Urea Lvonhilization For those proteins that are prepared in urea buffer, the protein is mixed with the matrix, vortexed many times, and then lyophilized. The lyophilized material may be used was is" for implants.
IN VIVO RAT BIOASSAY
Substantially pure BOP, BOP-rich extracts comprising protein having the properties set forth above, and several of the synthetic proteins have been incorporated in matrices to produce osteogenic devices, and assayed in rat for endochondral bone.
Studies in rats show the osteogenic effect to be dependent on the dose of osteogenic protein dispersed in the osteogenic device. No activity is observed if the matrix is implanted alone. The following sets forth guidelines for how the osteogenic devices disclosed herein might be assayed for determining active fractions of osteogenic protein when employing the isolation procedure of the invention, and evaluating protein constructs and matrices for biological activity.

A. Subcutaneous Implantation The bioassay for bone induction as described by Sampath and Reddi (Proc. Natl. Acad. Sci. USA
(1983) $Q: 6591-6595), _65- 1341610 1 is used to monitor the purification protocols for endochondral bone differentiation activity. This assay consists of implanting the test samples in subcutaneous sites in allogeneic recipient rats under ether anesthesia. Male Long-Evans rats, aged 28-32 days, were used. A vertical incision (1 cm) is made under sterile conditions in the skin over the thoraic region, and a pocket is prepared by blunt dissection. Approximately 25 mg of the test sample is implanted deep into the pocket and the incision is closed with a metallic skin clip. The day of implantation is designated as day of the experiment.
Implants were removed on day 12. The heterotropic site allows for the study of bone induction without the possible ambiguities resulting from the use of orthotopic sites.

B. Cellular Events The implant model in rats exhibits a controlled progression through the stages of matrix induced endochondral bone development including: (1) transient infiltration by polymorphonuclear leukocytes on day one; (2) mesenchymal cell migration and proliferation on days two and three; (3) chondrocyte appearance on days five and six; (4) cartilage matrix formation on day seven; (5) cartiliage calcification on day eight; (6) vascular invasion, appearance of osteoblasts, and formation of new bone on days nine and ten; (7) appearance of osteoblastic and bone remodeling and dissolution of _66- 1341610 1 the implanted matrix on days twelve to eighteen; and (8) hematopoietic bone marrow differentiation in the ossicle on day twenty-one. The results show that the shape of the new bone conforms to the shape of the implanted matrix.

C. Histological Evaluation Histological sectioning and staining is preferred to determine the extent of osteogenesis in the implants. Implants are fixed in Bouins Solution, embedded in parafilm, cut into 6-8 mm sections.
Staining with toluidine blue or hemotoxylin/eosin demonstrates clearly the ultimate development of endochondrial bone. Twelve day implants are usually sufficient to determine whether the implants show bone inducing activity.

D. Biological Markers Alkaline phosphatase activity may be used as a marker for osteogenesis. The enzyme activity may be determined spectrophotometrically after homogenization of the implant. The activity peaks at 9-10 days in vivo and thereafter slowly declines.
Implants showing no bone development by histology should have little or no alkaline phosphatase activity under these assay conditions. The assay is useful for quantitation and obtaining an estimate of bone formation very quickly after the implants are removed from the rat. Alternatively the amount of bone formation can be determined by measuring. the calcium content of the implant.

_67_ 1341610 1 Implants containing osteogenic protein at several levels of purity have been tested to determine the most effective dose/purity level, in order to seek a formulation which could be produced on a commercial scale. The results are measured by specific acivity of alkaline phosphatase, calcium content, and histological examination. As noted previously, the specific activity of alkaline phosphatase is elevated during onset of bone formation and then declines. On the other hand, calcium content is directly proportional to the total amount of bone that is formed. The osteogenic activity due to osteogenic protein is represented by "bone forming units". For example, one bone forming unit represents the amount of protein that is needed for half maximal bone forming activity as compared to rat demineralized bone matrix as control and determined by calcium content of the implant on day 12.
E. Results Dose curves are constructed for bone inducing activity in vivo at each step of the purification scheme by assaying various concentrations of protein. FIGURE 11 shows representative dose curves in rats as determined by alkaline phosphatase. Similar results are obtained when represented as bone forming units.
Approximately 10-12 jig of the Sephacryl-fraction, 3-4 - -1 pg of heparin-Sepharose-II fraction, 0.4-0.5 ug of the C-18 column purified fraction, and 20-25 ng of gel eluted highly purified 30 kD protein is needed for unequivocal bone formation (half maximum activity). 20-25 ng of the substantially pure protein per 25 mg of implant is normally sufficient to produce endochondral bone. Thus, 1-2 ng osteogenic protein per mg of implant is a reasonable dosage, although higher dosages may be used. (See section IB5 on specific activity of osteogenic protein.) OP1 expressed as set forth above (longer version), when assayed for activity histologically, induced cartilage and bone formation as evidenced by the presence of numerous chondrocytes in many areas of the implant and by the presence of osteoblasts surrounding vascular endothelium forming new matrix.

Deglycosylated xenogenic collagenous bone matrix (example: bovine) has been used instead of allogenic collagenous matrix to prepare osteogenic devices (see previous section) and bioassayed in rat for bone inducing activity in vivo. The results demonstrate that xenogenic collagenous bone matrix after chemical deglycosylation induces successful endochondral bone formation (Figure 19). As shown by specific activity of alkaline phosphotase, it is evident that the deglycosylated xenogenic matrix induced bone whereas untreated bovine matrix did not.

- -1 Histological evaluation of implants suggests that the deglycosylated bovine matrix not only has induced bone in a way comparable to the rat residue matrix but also has advanced the developmental stages that are involved in endochondral bone differentiation. Compared to rat residue as control, the HF treated bovine matrix contains extensively remodeled bone. Ossicles are formed that are already filled with bone marrow elements by 12 days. This profound action as elicited by deglycosylated bovine matrix in supporting bone induction is reproducible and is dose dependent with varying concentration of osteogenic protein.

ANIMAL EFFICACY STUDIES
Substantially pure osteogenic protein from bovine bone (BOP), BOP-rich osteogenic fractions having the properties set forth above, and several recombinant proteins have been incorporated in matrices to produce osteogenic devices. The efficacy of bone-inducing potential of these devices was tested in cat and rabbit models, and found to be potent inducers of osteogenesis, ultimately resulting in formation of mineralized bone. The following sets forth guidelines as to how the osteogenic devices disclosed herein might be used in a clinical setting.

A. Feline Model The purpose of this study is to establish a large animal efficacy model for the testing of the _70_ 1341610 1 osteogenic devices of the invention, and to characterize repair of massive bone defects and simulated fracture non-union encountered frequently in the practice of orthopedic surgery. The study is designed to evaluate whether implants of osteogenic protein with a carrier can-enhance the regeneration of bone following injury and major reconstructive surgery by use of this large mammal model. The first step in this study design consists of the surgical preparation of a femoral osteotomy defect which, without further intervention, would consistently progress to non-union of the simulated fracture defect. The effects of implants of osteogenic devices into the created bone defects were evaluated by the following study protocol.
A-1. Procedure Sixteen adult cats weighing less than 10 lbs. undergo unilateral preparation of a 1 cm bone defect in the right femur through a lateral surgical approach. In other experiments, a 2 cm bone defect was created. The femur is immediately internally fixed by lateral placement of an 8-hole plate to preserve the exact dimensions of the defect. There are three different types of materials implanted in the surgically created cat femoral defects: group I
(n = 3) is a control group which undergo the same plate fixation with implants of 4 M
guanidine-HC1-treated (inactivated) cat demineralized bone matrix powder (GuHC1-DBM) (360 mg); group II (n i I

_71_ 1341610 1 = 3) is a positive control group implanted with biologically active demineralized bone matrix powder (DBM) (360 mg); and group III (n = 10) undergo a procedure identical to groups I-II, with the addition of osteogenic protein onto each of the GuHCl-DBM
carrier samples. To summarize, the group III
osteogenic protein-treated animals are implanted with exactly the same material as the group II animals, but with the singular addition of osteogenic protein.
All animals are allowed to ambulate Ad libitum within their cages post-operatively. All cats are injected with tetracycline (25 mg/kg SQ each week for four weeks) for bone labelling. All but four group III animals are sacrificed four months after femoral osteotomy.

A-2. Radiomorphometrics In vivo radiomorphometric studies are carried out immediately post-op at 4, 8, 12 and 16 weeks by taking a standardized x-ray of the lightly anesthesized animal positioned in a cushioned x-ray jig designed to consistently produce a true anterio-posterior view of the femur and the osteotomy site. All x-rays are taken in exactly the same fashion and in exactly the same position on each animal. Bone repair is calculated as a function of mineralization by means of random point analysis. A
final specimen radiographic study of the excised bone is taken in two planes after sacrifice. X-ray -1 results are shown in FIGURE 12, and displaced as percent of bone defect repair. To summarize, at 16 weeks, 60% of the group III femors are united with average 86% bone defect regeneration. By contrast, the group I GuHCl-DMB negative-control implants exhibit no bone growth at four weeks, less than 10%
at eight and 12 weeks, and 16% ( 10%) at 16 weeks with one of the five exhibiting a small amount of bridging bone. The group II DMB positive-control implants exhibited 18% (f 3%) repair at four weeks, 35% at eight weeks, 50% (f 10%) at twelve weeks and 70% ( 12%) by 16 weeks, a statistical difference of p (0.01 compared to osteogenic protein at every month. One of the three (33%) is united at 16 weeks.
A-3. Biomechanics Excised test and normal femurs are immediately studied by bone densitometry, wrapped in two layers of saline-soaked towels, placed in two sealed plastic bags, and stored at -20 C until further study. Bone repair strength, load to failure, and work to failure are tested by loading to failure on a specially designed steel 4-point bending jig attached to an Instron testing machine to quantitate bone strength, stiffness, energy absorbed and deformation to failure. The study of test femurs and normal femurs yield the bone strength (load) in pounds and work to failure in joules. Normal femurs exhibit a strength of 96 ( 12) pounds. osteogenic protein-implanted femurs exhibited 35 ( 4) pounds, but when corrected for surface area at the site of 1 fracture (due to the "hourglass" shape of the bone defect repair) this correlated closely with normal bone strength. Only one demineralized bone specimen was available for testing with a strength of 25 pounds, but, again, the strength correlated closely with normal bone when corrected for fracture surface area.

A-4. Histomorphometry/Histology Following biomechanical testing the bones are immediately sliced into two longitudinal sections at the defect site, weighed, and the volume measured. One-half is fixed for standard calcified bone histomorphometrics with fluorescent stain incorporation evaluation, and one-half is fixed for decalcified hemotoxylin/eosin stain histology preparation.

A-5. Biochemistry Selected specimens from the bone repair site (n=6) are homogenized in cold 0.15 M NaCl, 3mM
NaHCO 3, pH 9.0 by a Spet freezer mill. The alkaline phosphatase activity of the supernatant and total calcium content of the acid soluble fraction of sediment are then determined.

A-6. Histopathology The final autopsy reports reveal no unusual or pathologic findings noted at necropsy of any of the animals studied. Portion of all major organs are * Trade-mark 1 preserved for further study. A histopathological evaluation is performed on samples of the following organs: heart, lung, liver, both kidneys, spleen, both adrenals, lymph nodes, left and right quadriceps muscles at mid-femur (adjacent to defect site in experimental femur). No unusual or pathological lesions are seen in any of the tissues. Mild lesions seen in the quadriceps muscles are compatible with healing responses to the surgical manipulation at the defect site. Pulmonary edema is attributable to the euthanasia procedure. There is no evidence of any general systemic effects or any effects on the specific organs examined.

A-7. Feline Study Summary The 1 cm and 2 cm femoral defect cat studies demonstrate that devices comprising a matrix containing disposed osteogenic protein can: (1) repair a weight-bearing bone defect in a large animal; (2) consistently induces bone formation shortly following (less than two weeks) implantation;
and (3) induce bone by endochondral ossification, with a strength equal to normal bone, on a volume for volume basis. Furthermore, all animals remained healthy during the study and showed no evidence of clinical or histological laboratory reaction to the implanted device. In this bone defect model, there was little or no healing at control bone implant sites. The results provide evidence for the successful use of osteogenic devices to repair large, non-union bone defects.

1 B. Rabbit Model:

B1. Procedure and Results Eight mature (less than 10 lbs) New Zealand White rabbits with epiphyseal closure documented by X-ray were studied. The purpose of this study is to establish a model in which there is minimal or no bone growth in the control animals, so that when bone induction is tested, only a strongly inductive substance will yield a positive result. Defects of 1.5 cm are created in the rabbits, with implantation of: osteogenic protein (n = 5), DBM (n = 8), GuHCl-DBM (n = 6), and no implant (n = 10). Six osteogenic protein implants are supplied and all control defects have no implant placed.

Of the eight animals (one animal each was sacrificed at one and two weeks), 11 ulnae defects are followed for the full course of the eight week study. In all cases (n = 7) following osteo-periosteal bone resection, the no implant animals establish no radiographic union by eight weeks. All no implant animals develop a thin "shell" of bone growing from surrounding bone present at four weeks and, to a slightly greater degree, by eight weeks.
In all cases (n = 4), radiographic union with marked bone induction is established in the osteogenic protein-implanted animals by eight weeks. As opposed to the no implant repairs, this bone repair is in the site of the removed bone.

1 Radiomorphometric analysis reveal 90%
osteogenic protein-implant bone repair and 18%
no-implant bone repair at sacrifice at eight weeks.
At autopsy, the osteogenic protein bone appears normal, while "no implant" bone sites have only a soft fibrous tissue with no evidence of cartilage or bone repair in the defect site.

B-2. Allograft Device In another experiment, the marrow cavity of the 1.5 cm ulnar defect is packed with activated osteogenic protein rabbit bone powder and the bones are allografted in an intercalary fashion. The two control ulnae are not healed by eight weeks and reveal the classic "ivory" appearance. In distinct contrast, the osteogenic protein-treated implants "disappear" radiographically by four weeks with the start of remineralization by six to eight weeks.
These allografts heal at each end with mild proliferative bone formation by eight weeks.

This type of device serves to accelerate allograph repair.

B-3. Summary These studies of 1.5 cm osteo-periosteal defects in the ulnae of mature rabbits show that: (1) it is a suitable model for the study of bone growth;
(2) "no implant" or GuHCl negative control implants yield a small amount of periosteal-type bone, but not _õ_ 1341610 1 medullary or cortical bone growth; (3) osteogenic protein-implanted rabbits exhibited proliferative bone growth in a fashion highly different from the control groups; (4) initial studies show that the bones exhibit 50% of normal bone strength (100% of normal correlated vol:vol) at only eight weeks after creation of the surgical defect; and (5) osteogenic protein-allograft studies reveal a marked effect upon both the allograft and bone healing.
The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims (126)

1. An isolated polynucleotide, or an isolated polynucleotide which is substantially identical to said polynucleotide, comprising a nucleic acid encoding a polypeptide comprising the following amino acid sequence: VPKPCCAPT, wherein said polypeptide, or a polypeptide which is substantially identical to said polypeptide, is capable of inducing cartilage and endochondral bone formation in a mammal when combined with a second polypeptide to produce a dimer, wherein said dimer is a homo-dimer or a hetero-dimer.

2. The polynucleotide according to claim 1, wherein said polypeptide comprises the following amino acid sequence:
LYVSFRDLGWQDWIIAPEGYAAYYCEGECAFPLNS
YMNATNHAIVQTLVHFINPETVPKPCCAPTQLNA
ISVLYFDDSSNVILKKYRNMVVRACGCH.

3. The polynucleotide according to claim 1, wherein said polypeptide comprises the following amino acid sequence:
HQRQACKKHELYVSFRDLGWQDWIIAPEGYAAYYCEGECAFPLNS
YNNATNHAIVQTLVHFINPETVPKPCCAPTQLNA
ISVLYFDDSSNVILKKYRNMVVRACGCH.

4. The polynucleotide according to any one of claims 1 to 3, wherein said polynucleotide comprises the nucleotide sequence as set forth in the bottom strand depicted in Figure 1B.

5. An isolated polynucleotide, or an isolated polynucleotide which is substantially identical to said polynucleotide, comprising a nucleic acid encoding a polypeptide comprising the following amino acid sequence: VPKPCCAPT, wherein said polypeptide, or a polypeptide which is substantially identical to said polypeptide, is other than OP1 and is capable of inducing cartilage and endochondral bone formation in a mammal when combined with a second polypeptide to produce a dimer, wherein said dimer is a homo-dimer or a hetero-dimer.

6. The polynucleotide according to any one of claims 1 to 4, wherein said polynucleotide comprises the nucleotide sequence as set forth in Figure 1A.

7. An isolated polynucleotide that is complementary to all or a portion of the nucleotide sequence as set forth in the bottom strand depicted in Figure 1B, wherein the polynucleotide is used as a probe.

8. An isolated polynucleotide that is complementary to all or a portion of the nucleotide sequence as set forth in Figure 1A, wherein the polynucleotide is used as a probe.

9. An isolated polynucleotide having a nucleotide sequence as set forth in Figure 13.

10. The isolated polynucleotide of any one of claims 1 to 4, wherein said polynucleotide is DNA.

11. The isolated polynucleotide of claim 5, wherein said polynucleotide is DNA.

12. The isolated polynucleotide according to claim 10, wherein said DNA is genomic DNA.

13. The isolated polynucleotide according to claim 11, wherein said DNA is genomic DNA.

14. The isolated polynucleotide according to claim 10, wherein said DNA is cDNA.

15. The isolated polynucleotide according to claim 11, wherein said DNA is cDNA.

16. The isolated polynucleotide of any one of claims 1 to 4, wherein said polynucleotide is RNA.

17. The isolated polynucleotide of claim 5, wherein said polynucleotide is RNA.

18. The isolated polynucleotide of any one of claims 1 to 4, wherein said polynucleotide is fused to a heterologous polynucleotide.

19. The isolated polynucleotide of claim 5, wherein said polynucleotide is fused to a heterologous polynucleotide.

20. The isolated polynucleotide of claim 18, wherein the heterologous polynucleotide encodes a heterologous regulatory sequence.

21. The isolated polynucleotide of claim 19, wherein the heterologous polynucleotide encodes a heterologous regulatory sequence.

22. A recombinant vector comprising the isolated polynucleotide of any one of claims 1, 2, 3, 4, 10, 12, 14, 16, 18 or 20.

23. A recombinant vector comprising the isolated polynucleotide of any one of claims 5, 6, 7, 8, 9, 11, 13, 15, 17, 19 or 21.

24. The vector according to claim 22, wherein the isolated polynucleotide is operably linked to a regulatory control sequence.

25. The vector according to claim 23, wherein the isolated polynucleotide is operably linked to a regulatory control sequence.

26. The vector according to claim 24, wherein said regulatory control sequence is a heterologous regulatory control sequence.

27. The vector according to claim 25, wherein said regulatory control sequence is a heterologous regulatory control sequence.

28. A host cell comprising the isolated polynucleotide of any one of claims 1, 2, 3, 4, 10, 12, 14, 16, 18 or 20.

29. A host cell comprising the isolated polynucleotide of any one of claims 5, 6, 7, 8, 9, 11, 13, 15, 17, 19 or 21.

30. The host cell according to claim 28, wherein the isolated polynucleotide is operably linked to a regulatory control sequence.

31. The host cell according to claim 29, wherein the isolated polynucleotide is operably linked to a regulatory control sequence.

32. The host cell according to claim 30, wherein said regulatory control sequence is heterologous to said polynucleotide.

33. The host cell according to claim 31, wherein said regulatory control sequence is heterologous to said polynucleotide.

34. A method of producing a host cell capable of expressing a polypeptide comprising genetically engineering cells with the vector of any one of claims 22, 24 or 26.

35. A method of producing a host cell capable of expressing a polypeptide comprising genetically engineering cells with the vector of any one of claims 23, 25 or 27.

36. The host cell produced by the method of claim 34.

37. The host cell produced by the method of claim 35.

38. A host cell comprising the isolated polynucleotide of any one of claims 5, 9, 11, 13, 15, 17, 19, or 21.

39. A method of producing a polypeptide, comprising expressing from the host cell of claim 38, the polypeptide encoded by said isolated polynucleotide, wherein said polypeptide is other than OP-1.

40. A method of producing a polypeptide comprising:
(1) culturing the host cell of claim 38, wherein the isolated polynucleotide is operably linked to a transcription promoter regulatory element, and whereby the isolated polynucleotide is expressed by said host cell to produce the polypeptide; and (2) recovering said polypeptide, wherein said polypeptide is other than OP-1.

41. The method according to claim 40, wherein said transcription promoter is heterologous to said polynucleotide.

42. A polypeptide produced by the method according to any one of claims 39, 40 or 41.

43. An isolated polypeptide, or a polypeptide which is substantially identical to said polypeptide, comprising the following amino acid sequence: VPKPCCAPT, wherein said polypeptide, or a polypeptide which is substantially identical to said polypeptide, is other than OP1 and is osteogenic.

44. The polypeptide according to claim 43, wherein said polypeptide is unglycosylated.

45. The polypeptide according to claim 43, wherein said polypeptide is glycosylated.

46. The polypeptide according to any one of claims 43 to 45, wherein said polypeptide is a dimer.

47. Use of the polypeptide, as defined by any one of claims 43 to 46, in the preparation of a medicament.

48. Use of the polypeptide, as defined by any one of claims 43 to 46, to stimulate osteogenesis in a patient in need of such therapy.

49. Use of the polypeptide encoded by the polynucleotide according to any one of claims 1 to 4, in the preparation of a medicament.

50. Use of the polypeptide encoded by the polynucleotide according to any one of claims 1 to 4, to stimulate osteogenesis in a patient in need of such therapy.

51. A method of making an osteogenic protein using the DNA
sequence of Figure 13, wherein said osteogenic protein comprises a pair of polypeptide chains disulfide bonded to produce a dimeric species which is a homo-dimer or a hetero-dimer, said dimeric species being capable of inducing cartilage and endochondral bone formation, at least one of said polypeptide chains comprising an amino acid sequence encoded by a nucleotide sequence which is the complement of a nucleotide sequence which specifically hybridizes with said DNA sequence of Figure 13, said method comprising expressing said nucleotide sequence which is the complement of a nucleotide sequence which specifically hybridizes with said DNA sequence of Figure 13.

52. A pharmaceutical composition for inducing endochondral bone formation, the composition consisting essentially of a) an osteogenic ingredient, and b) a pharmaceutically acceptable carrier, said osteogenic ingredient comprising a pair of polypeptide chains disulfide bonded in the unreduced state to form a dimeric species having a conformation such that the pair of polypeptide chains is capable of inducing cartilage and endochondral bone formation when disposed within a matrix and implanted in a mammal.

53. Osteogenic protein comprising a pair of unglycosylated polypeptide chains disulfide bonded to form a dimeric species having a conformation such that the pair of polypeptide chains is capable of inducing endochondral bone formation when disposed within a matrix and implanted in a mammal.

54. An osteogenic protein comprising a pair of polypeptide chains disulfide bonded to produce a dimeric species, said dimeric species being capable of inducing cartilage and endochondral bone formation when implanted in a mammal in association with a matrix, said protein having a half maximum bone inducing activity of about 25-50 ng/25 mg implant matrix.

55. The osteogenic protein according to claim 54, wherein at least one of said polypeptide chains comprises an amino acid sequence encoded by a nucleotide sequence which is the complement of a nucleotide sequence which specifically hybridizes in 1X SSPE, 0.1% SDS at 50°C
with the nucleotide sequence of Figure 13.

56. The composition of claim 52 having a half maximum bone inducing activity of about 25-50 ng/25 mg implant matrix.

57. The composition of claim 52 having a half maximum bone inducing activity of 0.8-1.0 ng/mg matrix.

58. The composition of claim 52 wherein said dimeric species has a molecular weight of about 30kD when oxidized, as determined by comparison to molecular weight standards in SDS-polyacrylamide gel electrophoresis.

59. The composition of claim 52 wherein said dimeric species has a molecular weight of about 27kD when oxidized, as determined by comparison to molecular weight standards in SDS-polyacrylamide gel electrophoresis.

60. The composition of claim 52 wherein one chain of said pair of polypeptide chains has an apparent molecular weight of about 18kD, and the other chain of said pair of polypeptide chains has an apparent molecular weight of about 16kD, both as determined after reduction by comparison to molecular weight standards in SDS-polyacrylamide gel electrophoresis.

61. The composition of claim 52 wherein one chain of said pair of polypeptide chains has an apparent molecular weight of about 16kD, and the other chain of said pair of polypeptide chains has an apparent molecular weight of about 14kD, both as determined after reduction by comparison to molecular weight standards in SDS-polyacrylamide gel electrophoresis.

62. An osteogenic device comprising: (a) a biocompatible, in vivo biodegradable matrix defining a scaffold of a dimension sufficient to permit attachment, proliferation and differentiation of migratory progenitor cells, and (b) substantially pure osteogenic protein disposed in said matrix and accessible to said cells, said protein comprising a pair of polypeptide chains disulfide bonded to produce a dimeric species having a conformation such that the pair of polypeptide chains is capable of inducing cartilage and endochondral bone formation in said mammal when disposed in said matrix and accessible to said cells.

63. The device of claim 62 wherein said matrix comprises close-packed particulate matter having a particle size within the range of 70 to 850 µm.

64. The device of claim 63 wherein said matrix comprises close-packed particulate matter having a particle size within the range of 70 to 420 µm.

65. The device of claim 62 wherein said matrix comprises:
(a) allogenic bone, or any of (b) demineralized, protein extracted, deglycosylated xenogenic bone, (c) demineralized, protein extracted, particulate xenogenic bone treated with HF or a protease, (d) materials selected from collagen, hydroxyapatite, calcium phosphates, and polymers comprising glycolic acid and/or lactic acid monomers, (e) a shape-retaining solid of loosely adhered particulate material, (f) a porous solid, (g) masticated muscle, (h) masticated tissue, or (i) a combination of any of (a) through (h).

66. The device of claim 62 wherein said osteogenic protein is unglycosylated.

67. The device of claim 62 wherein said osteogenic protein has a half maximum bone inducing activity of about 25-50 ng/25mg implant matrix.

68. The device of any one of claims 62 to 67 disposed within the cavity of allogenic bone.

69. The device of any one of claims 62 to 67 for use in inducing local cartilage or endochondral bone formation in a mammal, wherein the device is adapted to be implanted in a mammal at a locus accessible to migratory progenitor cells.

70. The device of any one of claims 62 to 67 for use in periodontal or dental reconstructive procedures.

71. The device of any one of claims 62 to 67 for use in craniofacial reconstructive procedures.

72. The device of any one of claims 62 to 67 for use in nonunion fractures.

73. The device of any one of claims 62 to 67 for use in cartilage repair, accelerating allograft repair or for treatment of osteoarthritis.

74. The composition of claim 52 for use in periodontal or dental reconstructive procedures.

75. The composition of claim 52 for use in craniofacial reconstructive procedures.

76. The composition of claim 52 for use in cartilage repair, accelerating allograft repair or for the treatment of osteoarthritis.

77. Osteogenic protein which comprises an amino acid sequence encoded by the sequence of Figure 1A, wherein said protein induces endochondral-bone formation when disposed within a matrix implanted in a mammal.

78. The composition of claim 52 having a half maximum bone inducing activity of 0.8-1.0 ng/mg matrix for use in periodontal or dental reconstructive procedures.

79. The composition of claim 52 having a half maximum bone inducing activity of 0.8-1.0 ng/mg matrix for use in craniofacial reconstructive procedures.

80. The composition of claim 52 having a half maximum bone inducing activity of 0.8-1.0 ng/mg matrix for use in cartilage repair, accelerating allograft repair or for the treatment of osteoarthritis.

81. The composition of claim 52 for use in heterotopic bone formation or repair of weight-bearing joints.

82. The device of any one of claims 62 to 67 for use in heterotopic bone formation or repair of weight-bearing joints.

83. The protein of any one of claims 53, 54, 55 or 77 for use in heterotopic bone formation or repair of weight-bearing joints.

84. The protein of any one of claims 53 to 55 having a half maximum bone inducing activity of 0.8-1.0 ng/mg matrix.

85. The protein of any one of claims 54 or 55 wherein said dimeric species has a molecular weight of about 30kD
when oxidized, as determined by comparison to molecular weight standards in SDS-polyacrylamide gel electrophoresis.

86. The protein of claim 53 wherein said dimeric species has a molecular weight of about 27kD when oxidized, as determined by comparison to molecular weight standards in SDS-polyacrylamide gel electrophoresis.

87. The protein of claim 54 or 55 wherein one chain of said pair of polypeptide chains has an apparent molecular weight of about 18kD, and the other chain of said pair of polypeptide chains has an apparent molecular weight of about 16kD, both as determined after reduction by comparison to molecular weight standards in SDS-polyacrylamide gel electrophoresis.

88. The protein of claim 53 wherein one chain of said pair of polypeptide chains has an apparent molecular weight of about 16kD, and the other chain of said pair of polypeptide chains has an apparent molecular weight of about 14kD, both as determined after reduction by comparison to molecular weight standards in SDS-polyacrylamide gel electrophoresis.

89. The protein of any one of claims 53 to 55 for use in periodontal or dental reconstructive procedures.

90. The protein of any one of claims 53 to 55 for use in craniofacial reconstructive procedures.

91. The protein of any one of claims 53 to 55 for use in cartilage repair, accelerating allograft repair or for the treatment of osteoarthritis.

92. The protein of any one of claims 53 to 55 having a half maximum bone inducing activity of 0.8-1.0 ng/mg matrix for use in periodontal or dental reconstructive procedures.

93. The protein of any one of claims 53 to 55 having a half maximum bone inducing activity of 0.8-1.0 ng/mg matrix for use in craniofacial reconstructive procedures.

94. The protein of any one of claims 53 to 55 having a half maximum bone inducing activity of 0.8-1.0 ng/mg matrix for use in cartilage repair, accelerating allograft repair or for the treatment of osteoarthritis.

95. Use of a therapeutically effective amount of the protein according to any one of claims 53 to 55 for induction of endochondral bone formation in a subject in need thereof.

96. Use of the protein according to any one of claims 53 to 55 in the manufacture of a pharmaceutical composition for induction of endochrondral bone formation.

97. Use of the device according to any one of claims 62 to 68 for induction of cartilage and bone formation.

98. Use of the device of any one of claims 62 to 67 for induction of local cartilage or endochondral bone formation in a mammal, wherein the device is adapted to be implanted in a mammal at a locus accessible to migratory progenitor cells.

99. Use of the device of any one of claims 62 to 67 in periodontal or dental reconstructive procedures.

100. Use of the device of any one of claims 62 to 67 in craniofacial reconstructive procedures.

101. Use of the device of any one of claims 62 to 67 in nonunion fractures.

102. Use of the device of any one of claims 62 to 67 in cartilage repair, accelerating allograft repair or for treatment of osteoarthritis.

103. Use of the device of any one of claims 62 to 67 in heterotopic bone formation or repair of weight-bearing joints.

104. Use of a therapeutically effective amount of the composition of claim 52 in periodontal or dental reconstructive procedures.

105. Use of a therapeutically effective amount of the composition of claim 52 in craniofacial reconstructive procedures.

106. Use of a therapeutically effective amount of the composition of claim 52 in cartilage repair, accelerating allograft repair or for the treatment of osteoarthritis.

107. Use of a therapeutically effective amount of the composition of claim 52 having a half maximum bone inducing activity of 0.8-1.0 ng/mg matrix in periodontal or dental reconstructive procedures.

108. Use of a therapeutically effective amount of the composition of claim 52 having a half maximum bone inducing activity of 0.8-1.0 ng/mg matrix in craniofacial reconstructive procedures.

109. Use of a therapeutically effective amount of the composition of claim 52 having a half maximum bone inducing activity of 0.8-1.0 ng/mg matrix in cartilage repair, accelerating allograft repair or for the treatment of osteoarthritis.

110. Use of a therapeutically effective amount of the composition of claim 52 in heterotopic bone formation or repair of weight-bearing joints.

111. Use of a therapeutically effective amount of the protein of any one of claims 53, 54, 55 or 77 in heterotopic bone formation or repair of weight-bearing joints.

112. Use of the protein of any one of claims 53, 54, 55 or 77 in the manufacture of a pharmaceutical composition for heterotopic bone formation or repair of weight-bearing joints.

113. Use a therapeutically effective amount of the protein of any one of claims 53 to 55 in periodontal or dental reconstructive procedures.

114. Use of the protein of any one of claims 53 to 55 in the manufacture of a pharmaceutical composition for use in periodontal or dental reconstructive procedures.

115. Use of a therapeutically effective amount of the protein of any one of claims 53 to 55 in craniofacial reconstructive procedures.

116. Use of the protein of any one of claims 53 to 55 in the manufacture of a pharmaceutical composition for use in craniofacial reconstructive procedures.

117. Use of a therapeutically effective amount of the protein of any one of claims 53 to 55 in cartilage repair, accelerating allograft repair or for the treatment of osteoarthritis.

118. Use of the protein of any one of claims 53 to 55 in the manufacture of a pharmaceutical composition for cartilage repair, accelerating allograft repair or for the treatment of osteoarthritis.

119. Use of a therapeutically effective amount of the protein of any one of claims 53 to 55 having a half maximum bone inducing activity of 0.8-1.0 ng/mg matrix for use in periodontal or dental reconstructive procedures.

120. Use of the protein of any one of claims 53 to 55 having a half maximum bone inducing activity of 0.8-1.0 ng/mg matrix in the manufacture of a pharmaceutical composition for use in periodontal or dental reconstructive procedures.

121. Use of a therapeutically effective amount of the protein of any one of claims 53 to 55 having a half maximum bone inducing activity of 0.8-1.0 ng/mg matrix in periodontal or dental reconstructive procedures.

122. Use of the protein of any one of claims 53 to 55 having a half maximum bone inducing activity of 0.8-1.0 ng/mg matrix in the manufacture of a pharmaceutical composition for use in periodontal or dental reconstructive procedures.

123. Use of a therapeutically effective amount of the protein of any one of claims 53 to 55 having a half maximum bone inducing activity of 0.8-1.0 ng/mg matrix in craniofacial reconstructive procedures.

124. Use of the protein of any one of claims 53 to 55 having a half maximum bone inducing activity of 0.8-1.0 ng/mg matrix in the preparation of a pharmaceutical composition for use in craniofacial reconstructive procedures.

125. Use of a therapeutically effective amount of the protein of any one of claims 53 to 55 having a half maximum bone inducing activity of 0.8-1.0 ng/mg matrix in cartilage repair, accelerating allograft repair or for the treatment of osteoarthritis.

126. Use of the protein of any one of claims 53 to 55 having a half maximum bone inducing activity of 0.8-1.0 ng/mg matrix in the manufacture of a pharmaceutical composition for cartilage repair, accelerating allograft repair or for the treatment of osteoarthritis.