Osteogenic devices

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, (3) methods of producing osteogenic proteins using recombinant DNA technology, and (4) osteogenically and chondrogenically active synthetic protein constructs.

BACKGROUND OF THE INVENTION 
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 synthetic forms of osteogenic 
protein, to a method of reproducibly purifying osteogenic protein from 
mammalian bone, to matrix materials which act as a carrier to induce 
osteogenesis in mammals, 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 chemotaxis of 
mesenchymal cells, proliferation of progenitor cells, differentiation of 
cartilage, vascular invasion, bone formation, remodeling, and finally 
marrow differentiation (Reddi (1981) Collagen Rel. Res. 1:209-226). 
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 78:7599-7603). 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 pure 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) 80). Urist et al. (Proc. Soc. Exp. Biol. Med. (1984) 
173:194-199) 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 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 Oct. 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 
purified to an essentially homogeneous state. This material is reported to 
induce bone formation when implanted into animals. 
International Application No. PCT/087/01537, published Jan. 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) 85: 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. 
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. 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. 
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 
native and non-native osteogenic proteins and methods for their production 
using recombinant DNA techniques. Another object is to provide novel 
biosynthetic forms of osteogenic proteins and a structural design for 
novel, functional osteogenic proteins. Another object is to provide a 
suitable deglycosylated collagenous bone matrix as a carrier for 
osteogenic protein for use in xenogenic implants. 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. 
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 or in the form of a biosynthetic construct. 
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. Investigation of the properties and structure 
of the native form osteogenic protein then permitted the inventors to 
develop a rational design for non-native forms, i.e., forms never before 
known in nature, capable of inducing bone formation. As far as applicants 
are aware, the constructs disclosed herein constitute the first instance 
of the design of a functional, active protein without preexisting 
knowledge of the active region of a native form nucleotide or amino acid 
sequence. 
A series of consensus DNA sequences were designed with the goal of 
producing an active osteogenic protein. The sequences were based on 
partial amino acid sequence data obtained from the natural source product 
and on observed homologies with unrelated genes reported in the 
literature, or the sequences they encode, having a presumed or 
demonstrated developmental function. Several of the biosynthetic consensus 
sequences have been expressed as fusion proteins in procaryotes, purified, 
cleaved, refolded, combined with a matrix, implanted in an established 
animal model, and shown to have endochondral bone-inducing activity. The 
currently preferred active totally biosynthetic proteins comprise two 
synthetic sequences designated COP5 and COP7. The amino acid sequences of 
these proteins are set forth below. 
##STR1## 
In these sequences and all other amino acid sequences disclosed herein, the 
dashes (--) are used as fillers only to line up comparable sequences in 
related proteins, and have no other function. Thus, amino acids 46-50 of 
COP7, for example, are NHAVV. Also, the numbering of amino acids is 
selected solely for purposes of facilitating Comparisons between 
sequences. Thus, for example, the DF residues numbered at 9 and 10 of COP5 
and COP7 may comprise residues, e.g., 35 and 36, of an osteogenic protein 
embodying invention. 
Thus, in one aspect, the invention comprises a protein comprising an amino 
acid sequence sufficiently duplicative of the sequence of COP5 or COP7 
such that it is capable of inducing endochondral bone formation when 
properlY folded and implanted in a mammal in association with a matrix. 
Some of these sequences induce cartilage, but not bone. Also, the bone 
forming materials may be used to produce cartilage if implanted in an 
avascular locus, or if an inhibitor to full bone development is implanted 
together with the active protein. Thus, in another aspect, the invention 
comprises a protein less than about 200 amino acids long in a sequence 
sufficiently duplicative of the sequence of COP5 or COP7 such that it is 
capable at least of cartilage formation when properly folded and implanted 
in a mammal in association with a matrix. 
In one preferred aspect, these proteins comprise species of the generic 
amino acid sequences: 
##STR2## 
where the letters indicate the amino acid residues of standard single 
letter code, and the Xs represent amino acid residues. Preferred amino 
acid sequences within the foregoing generic sequences are: 
##STR3## 
wherein each of the amino acids arranged vertically at each position in 
the sequence may be used alternatively in various combinations. Note that 
these generic sequences have 6 and preferably 7 cysteine residues where 
inter- or intramolecular disulfide bonds can form, and contain other 
critical amino acids which influence the tertiary structure of the 
proteins. These generic structural features are found in previously 
published sequences, none of which have been described as capable of 
osteogenic activity, and most of which never have been linked with such 
acitivity. 
Particular useful sequences include: 
##STR4## 
Vg1 is known Xenopus sequence heretofore not associated with bone 
formation. DPP is an amino acid sequence encoded by a drosophila gene 
responsible for development of the dorsoventral pattern. OP1 is a region 
of a natural sequence encoded by exons of a genomic DNA sequence retrieved 
by applicants. The CBMPs are amino acid sequences comprising subparts of 
mammalian proteins encoded by genomic DNAs and cDNAs retrieved by 
applicants. The COPs are biosynthetic protein sequences expressed by novel 
consensus gene constructs, designed using the criteria set forth herein, 
and not yet found in nature. 
These 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. As far as 
applicants are aware, the COP5 and COP7 constructs constitute the first 
instances of the design of a bioactive protein without preexisting 
knowledge of the active region of a native form nucleotide or amino acid 
sequence. 
The invention also provides native forms of osteogenic protein, extracted 
from bone or produced using recombinant DNA techniques. The substantially 
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 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) V--G--V--V--P--G--I--P--E--P--C--C--V--P--E; 
(6) V--D--F--A--D--I--G; 
(7) V--P--K--P--C--C--A--P--T; 
(8) D--E--Q--T--L--K--K--A--R--R--K--Q--W--I--?--P; 
(9) D--I--G--?--S--E--W--I--I--?--P; 
(10) S--I--V--R--A--V--G--V--V--P--G--I--P--E--P--?--?--V; 
(11) D--?--I--V--A--P--P--Q--Y--H--A--F--Y; 
(12) D--E--N--K--N--V--V--L--K--V--Y--P--N--M--T--V--E; 
(13) S--Q--T--L--Q--F--D--E--Q--T--L--K--?--A--R--?--K--Q; 
(14) 
D--E--Q--T--L--K--K--A--R--R--K--Q--W--I--E--P--R--N--?--A--R--R--Y--L; 
(15) A--R--R--K--Q--W--I--E--P--R--N--?--A--?--R--Y--?--?--V--D; and 
(16) R--?--Q--W--I--E--P--?--N--?--A--?--?--Y--L--K--V--D--?--A--?--?--G. 
The substantially pure (i.e., free of contaminating proteins having no 
osteoinductive activity) osteogenic proteins and the synthetics are useful 
in clinical applications in conjunction with a suitable delivery or 
support system (matrix). The matrix is made up of particles or 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 .mu.m, preferably 70-420 .mu.m. 
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 xenogenic 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 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. 
The consensus DNA sequences are also useful as probes for extracting genes 
encoding osteogenic protein from genomic and cDNA libraries. One of the 
consensus 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. 
##STR5## 
A longer active sequence is: 
##STR6## 
The probes have also retrieved the DNA sequences identified in 
PCT/087/01537, 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 leading to endochondral bone formation. 
Thus, in view of this disclosure, skilled genetic engineers can design and 
synthesize genes or 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 in native forms, truncated 
analogs, muteins, fusion proteins, and other constructs capable of 
inducing bone formation in mammals including humans. 
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 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. The other potential 
clinical application is in cartilage repair, for example, in the treatment 
of osteoarthritis.

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 known separation techniques, 
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 codons for the sequences, and observation of 
partial homology with known genes, also may be used as a probe. The probes 
may be used to isolate naturally occurring 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. 
The consensus sequence described above also may be refined by comparison 
with the sequences present in certain regulatory genes from drosophila, 
xenopus, and human followed by point mutation, expression, and assay for 
activity. This approach has been successful in producing several active 
totally synthetic constructs not found in nature (as far as applicants are 
aware) which have true osteogenic activity. 
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, 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 biosynthetics, 
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. 
I. NATURALLY SOURCED OSTEOGENIC PROTEIN 
A--PURIFICATION 
A1. Preparation of Demineralized Bone 
Demineralized bovine bone matrix is prepared by previously published 
procedures (Sampath and Reddi (1983) Proc Natl. Acad. Sci. USA 
80:6591-6595). 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.degree. 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 .mu.m 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 (75-425 .mu.m) from American Biomaterials) is then 
demineralized with 10 volumes of 0.5 N HCl at 4.degree. C. for 40 min., 
four times. Finally, neutralizing washes are done on the demineralized 
bone powder with a large volume of water. 
A2. Dissociative Extraction and Ethanol Precipitation 
Demineralized bone matrix thus prepared is dissociatively extracted with 5 
volumes of 4 M guanidine-HCl, 50 mM Tris-HCl, pH 7.0, containing protease 
inhibitors (5 mM benzamidine, 44 mM 6-aminohexanoic acid, 4.3 mM 
N-ethylmaleimide, 0.44 mM phenylmethylsulfonyfluoride) for 16 hr. at 
4.degree. 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.times.g for 10 min. 
at 4.degree. C.), and the supernatant is then subjected to ethanol 
precipitation. To one volume of concentrate is added five volumes of cold 
(-70.degree. C.) absolute ethanol (100%), which is then kept at 
-70.degree. C. for 16 hrs. The precipitate is obtained upon centrifugation 
at 10,000.times.g for 10 min. at 4.degree. C. The resulting pellet is 
resuspended in 4 l of 85% cold ethanol incubated for 60 min. at 
-70.degree. C. and recentrifuged. The precipitate is again resuspended in 
85% cold ethanol (2 l), incubated at -70.degree. C. for 60 min. and 
centrifuged. The precipitate is then lyophilized. 
A3. Heparin-Sepharose Chromatography I 
The ethanol precipitated, lyophilized, extracted crude protein is dissolved 
in 25 volumes of 6 M urea, 50 mM Tris-HCl, pH 7.0 (Buffer A) containing 
0.15 M NaCl, and clarified by centrifugation at 8,000.times.g for 10 min. 
The heparin-Sepharose is 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.degree. C. 
As shown in FIG. 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. Hydroxyapaptite-Ultrogel 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 500 mM 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 100 mM Na Phosphate 
(FIG. 2B). 
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 S-300 HR (High Resolution, 5 cm.times.100 cm column) is obtained 
from pharmacia and equilibrated with 4 M guanidine-HCl, 50 mM Tris-HCl, pH 
7.0. The bound protein fraction from HA-ultrogel is concentrated and 
exhanged from urea to 4 M guanidine-HCl, 50 mM Tris-HCl, 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-HCl, 50 mM 
Tris-HCl, 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 
A.sub.280 nm (FIG. 2C.). An aliquot of the individual fractions is be 
assayed for bone formation. Those fractions which have shown bone 
formation and have a molecular weigh less than 35 kD are pooled and 
concentrated via an Amicon ultrafiltration system with YM-10 membrane. 
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, 50 mM Tris-HCl, 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 (FIG. 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 4.degree. C. 
A7. Reverse Phase HPLC 
The protein is further purified by C-18 Vydac silica-based HPLC column 
chromatography (particle size 5 .mu.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 FIG. 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.degree. C. with a flow rate 
of 1.5 ml/min and 1.4 ml samples are collected in polycarbonate tubes. 
Protein is monitored by absorbance at A.sub.214 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. .sup.125 I-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 .sup.125 I-labelled protein. 
TABLE 1 
______________________________________ 
Elution of 30 kD Protein from SDS Gel 
% Eluted 
Buffer 0.5 mm 1.5 mm 
______________________________________ 
(1) dH.sub.2 O 22 
(2) 4M Guanidine-HCl, Tris-HCl, pH 7.0 
2 
(3) 4M Guanidine-HCl, Tris-HCl, pH 7.0, 
93 52 
0.5% Triton .times. 100 
(4) 0.1% SDS, Tris-HCl, pH 7.0 
98 
______________________________________ 
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 4 M 
guanidine-HCl containing 0.5% Triton.times.100 in Tris-HCl buffer or in 
Tris-HCl 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. 
In order to isolate substantially purified 30 kD or deglycosylated 27 kD 
protein for sequencing and characterization, the following steps are 
mentioned in Table 2. 
TABLE 2 
______________________________________ 
Preparation of Gel Eluted Protein 
(C-18 Pool or deglycoslated protein plus 
.sup.125 I-labelled 30 kD protein) 
______________________________________ 
1. Dry using vacuum centrifugation; 
2. Wash pellet with H.sub.2 O; 
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, 50 mM Tris-HCl, 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. 
______________________________________ 
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. In a 
separate experiment, a sample of gel eluted 30 kD protein is reduced, 
electrophoresed on an SDS gel, and transferred to an Immobilon membrane. 
The membrane is stained with Coomassie blue dye, cut into slices, and the 
slices are counted. Coomassie blue dye stains the 16 kD and 18 kD reduced 
species of the 30 kD protein almost exclusively. However, the counts 
showed significant smearing throughout the gel in addition to being 
concentrated in the 16 kD and 18 kD species. This suggests that the 
.sup.125 I-label can exhibit anomolous behavior on SDS gels and cannot be 
used as an accurate marker for cold protein under such circumstances. 
The yield is 0.5 to 1.0 .mu.g 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 .mu.g of gel eluted 30 kD protein (FIG. 3) is 
carboxymethylated and electrophoresed on an SDS-gel. The sample contains 
.sup.125 I-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. FIG. 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 .mu.g, 3-4 .mu.g, 
and 1-2 .mu.g, of protein respectively, as estimated by staining 
intensity. Prior to DSD 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. 
TABLE 3 
______________________________________ 
Isolation of the Subunits of the 30 kD protein 
(C-18 pool plus .sup.125 I 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 7.0. 
4. Concentrate and wash with H.sub.2 O in Centricon 
tube (10 kD membranes). 
5. Reduce and carboxymethylate in 1% SDS, 0.4M 
Tris, pH 8.5. 
6. Concentrate and wash with H.sub.2 O in Centricon 
tube. 
7. Electrophorese on SDS gel. 
8. Cut out the 16 kD and 18 kD subunits. 
9. Elute with 0.1% SDS, 50 mM Tris, pH 7.0. 
10. Concentrate and wash with H.sub.2 O in Centricon 
tubes. 
______________________________________ 
B. DEMONSTRATION THAT THE 30 KD PROTEIN IS OSTEOGENIC PROTEIN--BIOLOGICAL 
CHARACTERIZATION 
B1. Gel Slicing: 
Gel slicing experiments confirm that the isolated 30 kD protein is the 
protein responsible for osteogenic activity. 
Gels from the last step of the purification are sliced. Protein in each 
fraction is extracted in 15 mM Tris-HCl, pH 7.0 containing 0.1% SDS or in 
buffer containing 4 M guanidine-HCl, 0.5% non-ionic detergent 
(Triton.times.100), 50 mM Tris-HCl. The extracted proteins are desalted, 
concentrated, and assayed for endochondral bone formation activity. The 
results are set forth in FIG. 14. From this Figure it is clear that the 
majority of osteogenic activity is due to protein at 30 kD 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, 50 mM 
Tris-HCl, and is applied to a column of Con A-Sepharose equilibrated with 
the same buffer. The bound material is eluted in SDS Tris-HCl 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. 
B3. Gel Permeation Chromatography 
TSK-3000/2000 gel permeation chromatography in guanidine-HCl alternately is 
used to achieve separation of the high specific activity fraction obtained 
from C-18 chromatography (FIG. 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 
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 in 
vivo. The results of cleavage experiments demonstrate that pepsin 
treatment fails to destroy bone inducing capacity, whereas trypsin or CNBr 
completely abolishes the activity. 
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 is made without enzyme. The 
digestion mixture is incubated at room temperature for 16 hr. The digested 
product is then separated in 4 M guanidine-HCl 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. 
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 FIG. 17C), demonstrating 
that exposure to HF did not destroy the biological function of the 
protein. In addition, the specific activity of the deglycosylated protein 
is approximately the same as that of the native glycosylated protein. 
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 30 kD BOP. The 
results demonstrate that gel eluted substantially pure 30 kD 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 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 FIG. 14. Proteins 
are eluted from the slices in 4 M guanidine-HCl 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 substantially pure 30 kD osteogenic protein are 
implanted in varying concentrations in order to determine the half maximal 
bone inducing activity. 
FIG. 14 demonstrates that the bone inducing activity is due to proteins 
eluted at 28-34 kD region. The recovery of activity after the gel elution 
step is determined by calcium content. FIGS. 20A and 20B represent the 
bone inducing activity for the various concentrations of 30 kD protein 
before and after gel elution as estimated by calcium content. The 
concentration of protein is determined by gel scanning in the 30 kD 
region. 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 .mu.g of heparin sepharose I fraction, 
130-150 .mu.g of the HA ultrogel fraction, 10-12 .mu.g of the gel 
filtration fraction, 4-5 .mu.g of the heparin sepharose II fraction, 
0.4-0.5 .mu.g of the C-18 highly purified fraction, and 20-25 ng of gel 
eluted substantially purified 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. 
TABLE 4 
______________________________________ 
PURIFICATION OF BOP 
Biological 
Specific 
Purification 
Protein Activity Activity 
Purification 
Steps (mg.) Units* Units/mg. 
Fold 
______________________________________ 
Ethanol 30,000# 4,000 0.13 1 
Precipitate** 
Heparin 1,200# 2,400 2.00 15 
Sepharose I 
HA-Ultrogel 
300# 2,307 7.69 59 
Gel filtration 
20# 1,600 80.00 615 
Heparin 5# 1,000 200.00 1,538 
Sepharose II 
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. 
@ Proteins were measured by gel scanning method compared to known standar 
protein, bovine serum albumin. 
**Ethanolprecipitated 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 separate 
and used for bioassay. 
C. CHEMICAL CHARACTERIZATION OF BOP 
C1. 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 (FIG. 3A) 
and autoradiography. 
In order to extend the analysis of BOP, the protein was examined under 
reducing conditions. FIG. 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. Methods for the efficient 
elution of the proteins from SDS gels have been tested, and a protocol has 
been developed to achieve purification of both proteins. 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 possible 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 isoelectric focusing gel, using the 
iodinated 30 kD protein for detection. Using two dimensional gel 
electrophoresis and Con A for detection, the oxidized 30 kD protein show 
one species migrating 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 (FIG. 
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 (FIG. 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 .sup.125 
I-labelled 30 kD protein are incubated with the enzyme in the presence of 
SDS for 24 hours at 37.degree. C. As observed by SDS-PAGE, the treated 
samples appear as a prominent species at about 27 kD (FIG. 5B-1). Upon 
reduction, the 27 kD species is reduced to species having a molecular 
weight of about 14 kD-16 kD (FIG. 5B-2). 
Chemical cleavage of the carbohydrate moieties using hydrogen fluoride (HF) 
is performed to assess the role of carbohydrate on the bone inducing 
activity of BOP in vivo. Active osteogenic protein fractions pooled from 
the C-18 chromatography step are dried in vacuo over P.sub.2 O.sub.5 in a 
polypropylene tube, and 50 .mu.l freshly distilled anhydrous HF at 
-70.degree. C. is added. After capping the tube tightly, the mixture is 
kept at 0.degree. 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 
P.sub.2 O.sub.5 and KOH pellets. 
Following drying, the samples are dissolved in 100 .mu.l 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-HCl, 50 mM Tris-HCl, 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 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 presence 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.degree. 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 30 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 ammonium bicarbonate, pH 8.3. The pepsin 
reactions are done in 0.1% TFA. The digestion volume is 100 .mu.l and the 
ratio of enzyme to substrate is 1:10. .sup.125 I-labelled 30 kD osteogenic 
protein is added for detection. After incubation at 37.degree. C. for 16 
hr., digestion mixtures are dried down and taken up in gel sample buffer 
containing dithiothreitol for SDS-PAGE. FIG. 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 .mu.g) is fractionated 
by reverse-phase HPLC using a C-8 narrowbore column (13 cm.times.2.1 mm 
ID) with a TFA/acetonitrile gradient and a flow rate of 150 .mu.l/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 (FIG. 7A) are rechromatographed at least 
once under the same conditions in order to isolate single components 
satisfactory for sequence analysis. 
The HPLC profiles of the similarly digested 16 kD and 18 kD subunits are 
shown in FIGS. 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 .mu.g EndoAsp-N in 50 mM sodium phosphate 
buffer, pH 7.8 at 36.degree. 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 FIGS. 16A and 
16B. 
Various of the 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) V--G--V--V--P--G--I--P--E--P--C--C--V--P--E; 
(6) V--D--F--A--D--I--G; 
(7) VP--K--P--C--C--A--P--T; 
(8) D--E--Q--T--L--K--K--A--R--R--K--Q--W--I--?--P; 
(9) D--I--G--?--S--E--W--I--I--?--P; 
(10) S--I--V--R--A--V--G--V--P--G--I--P--E--P--?--?--V; 
(11) D--?--I--V--A--P--P--Q--Y--H--A--F--Y; 
(12) D--E--N--K--N--V--V--L--K--V--Y--P--N--M--T--V--E; 
(13) S--Q--T--L--Q--F--D--E--Q--T--L--K--?--A--R--?--K--Q; 
(14) 
D--E--Q--T--L--K--K--A--R--R--K--Q--W--I--E--P--R--N--?--A--R--R--Y--L; 
(15) A--R--R--K--Q--W--I--E--P--R--N--?--A--?--R--Y--?--?--V--D; and 
(16) R--?--Q--W--I--E--P--?--N--?--A--?--?--Y--L--K--V--D--?--A--?--?--G. 
C6. Amino Acid Analysis 
Strategies for obtaining amino acid composition were developed using gel 
elution from 15% SDS gels, transfer onto Immobilon, and hydrolysis. 
Immobilon membrane is a polymer of vinylidene difluoride and, therefore, 
is not susceptible to acid cleavage. 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 analysis as described below. 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. 
Samples are run on 15% SDS gels, transferred to Immobilon, and stained with 
Coomassie blue. The bands of interest are excised from the Immobilon, with 
a razor blade and placed in a 6.times.50 mm Corning test tube cleaned by 
pyrolysis at 550.degree. C. When cysteine is to be determined, the samples 
are treated with performic acid, which converts cysteine to cysteic acid. 
Cysteic acid is stable during hydrolysis with HCl, and can be detected 
during the HPLC analysis by using a modification of the normal Pico-Tag 
eluents (Millipore) and gradient. The performic acid is made by mixing 50 
.mu.l 30% hydrogen peroxide with 950 .mu.l 99% formic acid, and allowing 
this solution to stand at room temperature for 2 hr. The samples are then 
treated with performic acid (PFA); 20 .mu.l PFA is pippetted onto each 
sample and placed in an ice bath at 4.degree. C for 2.5 hours. After 2.5 
hr. the PFA is removed by drying in vacuo, and the samples are then 
hydrolyzed. A standard protein of known composition and concentration 
containing cysteine is treated with PFA and hydrolyzed concurrently with 
the osteogenic protein samples, to take as a control for hydrolysis and 
amino acid chromatography. 
The hydrolysis of the osteogenic protein samples is done in vacuo. The 
samples, with empty tubes and Immobilon blanks, are placed in a hydrolysis 
vessel which is placed in a dry ice/ethanol bath to keep the HCl from 
prematurely evaporating. 200 .mu.l 6 N HCl containing 2% phenol and 0.1% 
stannous chloride are added to the hydrolysis vessel outside the tubes 
containing the samples. The hydrolysis vessel is then sealed, flushed with 
prepurified nitrogen, evacuated, and then held at 115.degree. C. for 24 
hours, after which time the HCl is removed by drying in vacuo. 
After hydrolysis, each piece of Immobilon is transferred to a fresh tube, 
where it is rinsed twice with 100 .mu.l 0.1% TFA, 50% acetonitrile. The 
washings are returned to the original sample tube, which is then redried 
as below. A similar treatment of amino acid analysis on Immobilon can be 
found in the literature (LeGendre and Matsudaira (1988) Biotechniques 
6:154-159). 
The samples are redried twice using 2:2:1 ethanol:water:triethylamine and 
allowed to dry at least 30 min. after each addition of redry reagent. 
These redrying steps bring the sample to the proper pH for derivatization. 
The samples are derivatized using standard methodology. The solution is 
added to each sample tube. The tubes are placed in a desiccator which is 
partially evacuated, and are allowed to stand for 20 min. The desiccator 
is then fully evacuated, and the samples are dried for at least 3 hr. 
After this step the samples may be stored under vacuum at -20.degree. C. 
or immediately diluted for HPLC. The samples are diluted with Pico-Tag 
Sample Diluent (generally 100 .mu.l) and allowed to stand for 20 min., 
after which they are analyzed on HPLC using the Pico Tag chromatographic 
system with some minor changes involving gradients, eluents, initial 
buffer conditions and oven temperature. 
After HPLC analysis, the compositions are calculated. The molecular weights 
are assumed to be 14.4 kD, 16.2 kD, and 27 kD to allow for 10% 
carbohydrate content. The number of residues is approximated by dividing 
the molecular weight by the average molecular weight per amino acid, which 
is 115. The total picomoles of amino acid recovered is divided by the 
number of residues, and then the picomoles recovered for each amino acid 
is divided by the number of picomoles per residue, determined above. This 
gives an approximate theoretical number of residues of each amino acid in 
the protein. Glycine content may be overestimated in this type of 
analysis. 
Composition data obtained are shown in TABLE 5. 
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 
______________________________________ 
*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, Mass.), 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-HCl extract. 
One third of the ethanol precipitate (0.5 g) is used for gel filtration 
through 4 M guanidine-HCl (FIG. 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 is eluted stepwise by using the same 
buffer containing 100 mM and 500 mM phosphate (FIG. 10B). Bioassay of HAP 
bound and unbound fractions demonstrates that only the fraction eluted by 
100 mM 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 (FIG. 10D. FIG. 10C describes 
the elution profile for the intervening gel filtration step described 
above.) 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 
E-1 PROBE DESIGN 
A synthetic consensus gene shown in FIG. 13 was designed as a hybridization 
probe (and to encode a consensus protein, see below) 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. 
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 Xenopus 
VG1 protein, and somewhat less homology to inhibin and TGF-beta, as 
demonstrated below in TABLE 6. 
TABLE 6 
______________________________________ 
protein amino acid sequence homology 
______________________________________ 
(BOP) (DPP) 
##STR7## (9/15 matches) 
(BOP) (Vgl) 
##STR8## (6/15 matches) 
(BOP) (inhibin) 
##STR9## (5/15 matches) 
(BOP) (TGF-beta) 
##STR10## (4/15 matches) 
(BOP) (Vgl) 
##STR11## (12/20 matches) 
(BOP) (inhibin) 
##STR12## (12/20 matches) 
(BOP) (TGF-beta) 
##STR13## (6/19 matches) 
(BOP) (DPP) 
##STR14## (12/20 matches) 
(BOP) (DPP) 
##STR15## (5/5 matches) 
(BOP) (Vgl) 
##STR16## (4/5 matches) 
(BOP) (TGF-beta) 
##STR17## (4/5 matches) 
(BOP) (inhibin) 
##STR18## (2/4 matches) 
______________________________________ 
*match 
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 Vg1 are used; (4) If Vg1 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 Vg1 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 COP0, (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. 
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 COP0 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 (COP0) 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 
5.times.SSPE, 10.times.Denhardt's mix, 0.5% SDS at 50.degree. 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 
1.times.SSPE with 0.1% SDS and twice at 50.degree. C. for 2.times.30 min. 
in the same solution. Using this procedure, twenty-four positive clones 
were found. Two of these yielded the genes corresponding to BMP-2b, one 
yielded BMP-3 (see PCT U.S. No. 87/01537) and two contained a gene never 
before reported designated OP1, osteogenic protein-1 described below. 
Southern blot analysis of lambda #13 DNA showed that an approximately 3 kb 
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 
COP0 probe. This showed that a 1 kb (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. 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 4 kb 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: 
##STR19## 
A longer active sequence is: 
##STR20## 
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 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. 
II. RECOMBINANT NON-NATIVE OSTEOGENIC PROTEIN CONSTRUCTS 
A. Protein Design 
This section discloses the production of novel recombinant proteins capable 
of inducing cartilage and endochondral bone comprising a protein structure 
duplicative of the functional domain of the amino acid sequence encoded by 
consensus DNA sequences derived from a family of natural proteins 
implicated in tissue development. These gene products/proteins are known 
to exist in active form as dimers and are, in general, processed from a 
precursor protein to produce an active C-terminal domain of the precursor. 
The recombinant osteogenic/chondrogenic proteins are "novel" in the sense 
that, as far as applicants are aware, they do not exist in nature or, if 
they do exist, have never before been associated with bone or cartilage 
formation. The approach to design of these proteins was to employ amino 
acid sequences, found in the native isolates described above, in 
polypeptide structures which are patterned after certain proteins reported 
in the literature, or the amino acid sequences inferred from DNAs reported 
in the literature. Thus, using the design criteria set forth above in the 
probe design section, and refining the amino acid sequence as more protein 
sequence information was learned, a series of synthetic proteins were 
designed with the hope and intent that they might have osteogenic or 
chondrogenic activity when tested in the bioassay system disclosed below. 
It was noted, for example, that DPP from drosophila, VG1 from Xenopus, the 
TGF beta family of proteins, and to a lesser extent, alpha and beta 
inhibins, had significant homologies with certain of the sequences derived 
from the naturally sourced OP product. (FIG. 18.) Study of these proteins 
led to the realization that a portion of the sequence of each had a 
structural similarity observable by analysis of the positional 
relationship of cysteines and other amino acids which have an important 
influence on three dimensional protein conformation. It was noted that a 
region of these sequences had a series of seven cysteines, placed very 
nearly in the same relative positions, and certain other amino acids in 
sequence as set forth below: 
##STR21## 
wherein each X independently represents an amino acid. Expression 
experiments with constructs patterned after this template amino acid 
sequence showed activity occurred with a shorter sequence having only six 
cysteines: 
##STR22## 
wherein each X independently represents an amino acid. Within these 
generic structures are a multiplicity of specific sequences which have 
osteogenic or chondrogenic activity. Preferred structures are those having 
the amino acid sequence: 
##STR23## 
wherein, in each position where more than one amino acid is shown, any one 
of the amino acids shown may be used. Novel active proteins also are 
defined by amino acid sequences comprising an active domain beginning at 
residue number 6 of this sequence, i.e, omitting the N terminal CXXXX, or 
omitting any of the preferred specific combinations such as CKRHP, CRRKQ, 
CKRHE, etc, resulting in a construct having only 6 cysteine residues. 
After this work, PCT 87/01537 was published, and it was observed that the 
proteins there identified as BMPII a and b and BMPIII each comprised a 
region embodying this generic structure. These proteins were not 
demonstrated to be osteogenic in the published application. However, 
applicants discovered that a subpart of the amino acid sequence of these 
proteins, properly folded, and implanted as set forth herein, is active. 
These are disclosed herein as CBMPIIa, CBMPIIb, and CBMPIII. Also, the OP1 
protein was observed to exhibit the same generic structure. 
Thus, the preferred osteogenic proteins are expressed from recombinant DNA 
and comprise amino acid sequences including any of the following 
sequences: 
##STR24## 
As shown in FIG. 18, these sequences have considerable homology with the 
alpha and beta inhibins, three forms of TGF beta, and MIS. 
B. Gene Preparation 
The synthetic genes designed as described above preferably are produced by 
assembly of chemically synthesized oligonucleotides. 15-100 mer 
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. Natural 
gene sequences and cDNAs also may be used for expression. 
C. Expression 
The genes can be expressed in appropriate prokaryotic hosts such as various 
strains of E. coli. For example, if the gene is to be expressed in E. 
coli. it must first be cloned into an expression vector. An expression 
vector (FIG. 21A) based on pBR322 and containing a synthetic trp promoter 
operator and the modified trp LE leader can be opened at the EcoRI and 
PSTI restriction sites, and a FB-FB COP gene fragment (FIG. 21B) can be 
inserted between these sites, where FB is fragment B of Staphylococcal 
Protein A. The expressed fusion protein results from attachment of the COP 
gene to a fragment encoding FB. The COP 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 COP protein. 
D. Production of Active Proteins 
The following procedure was followed for production of active recombinant 
proteins. E. coli cells containing the fusion proteins were lysed. The 
fusion proteins were purified by differential solubilization. In the case 
of the COP 1, 3, 4, 5, and 7 fusion proteins, cleavage was with dilute 
acid, and the resulting cleavage products were passed through a 
Sephacryl-200HR column. The Sephacryl column separated most of the 
uncleaved fusion products from the COP 1, 3, 4, 5, and 7 analogs. In the 
case of the COP 16 fusion protein, cleavage was with a more concentrated 
acid, and an SP-Trisacryl column was used to separate COP 16, the leader 
protein, and the residual fusion protein. The COP fractions from any of 
the COP analogs were then subjected to HPLC on a semi-prep C-18 column. 
The HPLC column primarily separated the leader proteins and other minor 
impurities from the COP analogs. 
Initial conditions for refolding of COP analogs were at pH 8.0 using Tris, 
GuHCl, dithiothreitol. Final conditions for refolding of COP analogs were 
at pH 8.0 using Tris, oxidized glutathione, and lower amounts of GuHCl and 
dithiothreitol. 
E. Production of Antisera 
Antisera to COP 7 and COP5 were produced in New Zealand white rabbits. 
Western blots demonstrate that the antisera react with COP 7 and COP5 
preparations. Antisera to COP 7 has been tested for reactivity to bovine 
osteogenic protein samples. Western blots show a clear reaction with the 
30 kD protein and, when reduced, with the 16 kD subunit. The 
immunoreactive species appears as a closely-spaced doublet in the 16K 
subunit region, similar to the 16K doublet seen in Con A blots. 
III. MATRIX PREATION 
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 .mu.m 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 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. 
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 Allogenic 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 .mu.m sieve. The bone particles are subjected to 
dissociative extraction with 4 M guanidine-HCl. 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 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), while 
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. 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. 
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 .mu.M are collected. The sample is dried in vacuo over P.sub.2 
O.sub.5, transferred to the reaction vessel and anhydrous hydrogen 
fluoride (HF) (10-20 ml/g of matrix) is then distilled onto the sample at 
-70.degree. C. The vessel is allowed to warm to 0.degree. 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) 1 g/200 ml and stir at 4.degree. 
C. for 2 hrs; 
(2) centrifuge then treated again with TBS, 1 g/200 ml and stir at 
4.degree. C. overnight; and 
(3) centrifuged; discard supernatant; water wash residue; and then 
lyophilized. 
IV. FABRICATION OF 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-HCl. 
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 Lyophilization 
In this procedure, osteogenic protein in an acetonitrile trifluroacetic 
acid (ACN/TFA) solution was added to the carrier. Samples were vigorously 
vortexed many times and then lyophilized. Osteogenic protein was added in 
varying concentrations obtained at several levels of purity that have been 
tested to determine the most effective dose/purity level in rat in vivo 
assay. C. Urea Lyophilization 
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 "as is" for implants. 
V. 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) 80: 6591-6595), herein incorporated by 
reference, 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 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. In order to estimate the amount of bone formation, the 
calcium content of the implant is determined. 
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 an industrial scale. The 
results are measured by specific activity of alkaline phosphatase and 
calcium content, and histological examination. 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 
E-1. Natural Sourced Osteogenic Protein 
Dose curves are constructed for bone inducing activity in vivo at each step 
of the purification scheme by assaying various concentrations of protein. 
FIG. 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 .mu.g of the Sephacryl-fraction, 3-4 .mu.g of 
heparin-Sepharose-II fraction, 0.4-0.5 .mu.g 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 
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.) 
E-2. Xenogenic Matrix Results 
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 xenogehic collagenous bone matrix after 
chemical deglycosylation induces successful endochondral bone formation 
(FIG. 19). As shown by specific activity of alkaline phosphatase, it is 
evident that the deglycosylated xenogenic matrix induced bone whereas 
untreated bovine matrix did not. 
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. 
E-3. Synthetic/Recombinant Proteins (COP5, COP7) 
The device that contained only rat carrier showed complete absence of new 
bone formation. The implant consists of carrier rat matrix and surrounding 
mesenchymal cells. Again, the devices that contained rat carrier and not 
correctly folded (or biologically inactive) recombinant protein also 
showed complete absence of bone formation. These implants are scored as 
cartilage formation (-) and bone formation (-). The endochondral bone 
formation activity is scored as zero percent (0%). (FIG. 22A) 
Implants included that biologically active recombinant protein, however, 
showed evidence of endochondral bone formation. Histologically they showed 
new cartilage and bone formation. 
The cartilage formation is scored as (+) by the presence of 
metachromatically stained chondrocytes in center of the implant, as (++) 
by the presence of numerous chondrocytes in many areas of the implant and 
as (+++) by the presence of abundant chondrocytes forming cartilage matrix 
and the appearance of hypertrophied chondrocytes accompanying cartilage 
calcification (FIG. 22B). 
The bone formation is scored as (+) by the presence of osteoblast 
surrounding vascular endothelium forming new matrix, and as (++) by the 
formation of bone due to osteoblasts (as indicated by arrows) and further 
bone remodeling by the appearance of osteoblasts in apposition to the rat 
carrier. Vascular invasion is evident in these implants (FIG. 22B). 
The overall bone inducing activity due to recombinant protein is 
represented as percent response of endochondral bone formation (see Table 
7 below). The percent response means the area of the implant that is 
covered by newly induced cartilage and bone as shown by histology in low 
magnification. 
TABLE 7 
______________________________________ 
HISTOLOGICAL EVALUATION OF RECOMBINANT 
BONE INDUCTIVE PROTEINS 
Percent 
Implanted Cartilage Bone Response in 
Protein Formation Formation the Implant 
______________________________________ 
COP-5 +++ ++ 15% 
COP-5 ++ + 5% 
COP-7 +++ ++ 30% 
COP-7 +++ ++ 20% 
COP-7 ++ + 20% 
COP-7 ++ + 10% 
COP-7 +++ ++ 30% 
COP-7 ++ ++ 20% 
COP-5 +++ ++ 20% 
______________________________________ 
VI. ANIMAL EFFICACY STUDIES 
Substantially pure osteogenic protein from bovine bone (BOP), BOP-rich 
osteogenic fractions having the properties set forth above, and several of 
the synthetic/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 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 p reparation 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-HCl-treated (inactivated) 
cat demineralized bone matrix powder (GuHCl-DBM) (360 mg); group II (n=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 I 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 results are shown in FIG. 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% 
(.+-.3%) repair at four weeks, 35% at eight weeks, 50% (.+-.10%) at twelve 
weeks and 70% (.+-.12%) by 16 weeks, a statistical difference of p&lt;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.degree. 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 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-5Biochemistry 
Selected specimens from the bone repair site (n=6) are homogenized in cold 
0.15 M NaCl, 3 mM NaHCO.sub.3, pH 9.0 by a Spex 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 
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. 
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. 
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 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.