Note: Descriptions are shown in the official language in which they were submitted.
g~ L3~i
P TI~LLY PU~IFIED ~ONE-INDUCING FACTOR
Descrip~io~:
Technical Field
The p~e6ent invention relate6 to protein
che~istry and osteoplasty. ~ore particulaLly, it
relates to a partially purified pro~einaceous factor
that promote6 rapid bone growth.
Backqround ~rt
It has been e6tabli6hed ~hat hone contain6
material6 which can 6timulate the formation of new bone
when placed in contact with living system6. ~Urist, M.
R., Clin OrthoP ~1968) 56:37 Science (1965) 150:893;
Reddi, ~. H., et al, Proc Natl ~cad Sci (~SA) (1972~
69:1601.) ~ttempts have been made to puLify whatever
factors are res~onsibla for this activity. ~ "bone
morphogenic pLotein" (BMP) was extracted from
deminaralized bone using urea or guanidine hydrochloride
and reprecipitated according to the disclo~ures in U.S.
Patent Nos. 4,294,753 and 4,455,256 to Urist. Urist
subsequently reported (Uris~, M. R., Clin Orthop Rel Res
(1982) 16 :219) that ion exchange purification of thi6
crude protein mixture yielded an activity which wa6
unad60rbed to carboxymethyl cellulose (CMC) at pH 4.~.
Urist'6 most recent report~ (Science (1983) 220:680-685
and Proc ~atl ~cad Science (US~) (1984) 81:371-375)
describe BMPs having molecular weights of 17,500 and
18,500 dal~ons.
U.S. 4~434,094 repoLted the partial
purification of what i6 evidently a bone
generation-stimulating, bone-derived protein by
extraction with chaotropic agents, fractionation on
~66~;~S
anion and cation exchange columns, and recovery of the
activity from a fraction ad~orbed to CMC at pH 4.8.
This new protein fraction W,l~ termed "osteogenic factor"
(OF) and was characterized as having a molecular weight
below about 30,000 daltons and as tracking the
purification proce6~ de6cribed. Two proteins were
purified to homogeneity frol~ this protein. Those two
proteins eluted from CMC re6in at about a 150-200 mM
NaCl gradient and have molular weights of about 26,000
daltons. These proteins are described in commonly owned
European Patent Application 85.304348.6, publi6hed 22
January 1986. While these proteins are believed to be
involved in osteogenesis, they exhibit no osteogenic
activity by themselves.
The present application concerns a bone
inducing factor present in the above-described CMC-bound
f~action that elutes from the re6in in the portion of
the NaCl gradient below about 150 mM.
20 Disclosure of the Invention
The invention relates to methods for obtaining
a partially purified bone inducing factor from
demineralized bons (DMB), the factor prepared thereby,
implant material6 con~aining the factor, and methods for
inducing bone formation in mammal~ u6ing the factor.
The proces6 for preparing the partially
purified bone-inducing factor compri~e6:
~ a) extracting demineralized bone with a
chaotropic Sdis60ciative) extractant that solubilize6
nonfibrous proteins
~ b3 6ubjecting the extract from 6tep (a) to gel
filtration to recover a fraction containing proteins of
molecular weight 10,000-30,000 daltons:
~266~35
(c) adsoebing the Eraction from step (b~ onto a
carboxymethyl cellulose cation exchanger at
approximately pH ~.5-5~5; a:nd
(d) eluting an active protein fraction from the
cation exchanger with a sodium chlori~e gradient
charac~eri~ed in that this active fcaction is eluted in
the por~ion of the gLadient from about 10 mM to about
150 mM and in the presence of a nonionic chaotropic
agent.
The partially purified bone-inducing factor is
characterized in that it is p~epared by the
above-described process.
The osteogenic implant material is
characterized in that its active ingredient is the
lS above-described partially purified bone-inducing factor.
The method of inducing bone formation at a
predetermined site in a living mammal comprises
implanting an osteogenic material at said site and i6
characterized in that the material i6 the
above~described partially purified bone-inducing factor.
Brief Description of the Drawinqs
In the drawings:
Figure 1 is a graph of the optical densities
~absorbances~ (2B0 nm) of the gel filtration fractions
of the example (1~C),
Figure 2 is a graph of the op~ical densities
~2eo nm) of eluate fraction~ from the preparative ibn
exchange chromatography of the example (1~D)
Yigure6 3ta) and 3(b) are graph~ 6howing the
speci~ic chondrogenic activity as determined per 1~F of
certain ~rotein6 described in the example;
~Z~643~
Figure 4 is a 6chematic diagram outlining ~he
reconstitution procedure de~;cribed in ~G of the example,
and
Figure 5 is a table that provide6 details of
the reconstituted ~ample6 de6cribed in 1~G and their
specific chondlogenic activiLty as determined by the
assay of 1~F.
Modes of Carryinq Out the Invention
The native sources of the bone-inducing fator
of t~e claimed invention are bone, dentin,
osterosarcomas or chondrosarcomas. In view of the
showing that bone inductive proteins from human, monkey,
bovine and rat are nonspecies specific in their ability
to produce endochondral bone in xenogeneic implants
(Sampath, T. K., et al, Proc Natl Acad Sci (~SA) (1983)
80:6591) it i6 believed that the factor of the claimed
invention has been highly conserved among mammalian
species ~i.e., fac~ors from different mammalian 6pecies
20 will have sub6tantially homologous amino acid sequences
that vary, if at all, in one or more amino acid residue
additions, deletions, or substi~utions t~at do not
affect the nonspecies specific bone inducing activity of
the molecule adversely). In this regard the term6
~substantially equivalent~ and "sub6tantially
homologousl' as used herein are intended to mean factor~
regardle~s of species, that have the same amino acid
composition or sequence, as the case may be, of the
factor described in the example and factor~ of similar
but dif~erent amino acid compo&ition or sequence, which
difference(s) does not affect nonspecies ~ecific
endochondral bone-inducing activity adversaly.
Accordingly, such factors may be derived from cell~ or
~ ue of diverse mammalian origin. The source of
~2~ 3S
factor prepared by purification from native ~ources is
advantageously porcine or bovine long bone because of
its ready availability.
A variety of initial preparation procedure6 are
possible, but basically the bone i8 first cleaned u6ing
mechanical or abra6ive techniques, fragmented, and
further wa6hed with, for example, dilute aqueou6 acid
preferably at low temperatuLe, and then defatted by
extraction with a lipophilic solvent such a6 ether or
ethyl acetate. The bone is then demineralized by
removal of the calcium phosphates in their various
forms, usually by extraction with strongeL acid. The6e
technique~ are understood in the art, and are disclosed,
for example, in ~.S. 4,434,094. The resulting
prepa~ation, a demineralized bone, is the starting
material for the ereparation of the claimed osteogenic
factor.
The initial extraction is designed to remove
the nonfibrous te.g., noncollagenous) proteins f~om the
demineralized bone. This can be done with the use of
chaotropic agents such as guanidine hydrochloride (at
least about 4 molar), urea t8 molar) plus salt, or
sodium dodecyl6ulfate (at least about 1~ by volume) or
6uch other chao~ropic agent6 as are known in the art
(Termine, et al, J Biol Chem (1980~ 255:97~0-9772; and
Sajera and Hascall, J Biol Chem (1969) 244:77-87 and
2334-2396). The extraction i8 preferably carried out at
reduced temperatures in the presence of a protea~e
inhibitor to reduce the likelihood of digestion or
denaturation of the extracted protein. ~xamples of
protea6e inhibitors that may be included are
phenylmsthyl6ulfonylfIuoride (PMSF) sodium azide,
N-ethyl maleimide (NEM), benzamidine, and 6-amino
hexanoic acid. The pH of the medium depend6 upon the
~6~i4;~S
axtractant selected. The proce~s of extraction
generally takes on the order of about 4 hr to 1 day.
After extraction, the extractant may be removed
by suitable means such as dialysis against water,
preceded by concentration by ultrafiltration if
desired. Salts can also be removed by controlled
electrophoresis, or by molecular 6ieving, or by any
other means known in the art. It iB alBO preferred to
maintain a low temperature during this process BO afi to
minimize denaturation of the protein6. Alternatively,
the extractant chaotropic agent need not be ~emoved, but
rather the solution need only be concentrated, for
example, by ultrafiltration.
The extract, dissolved or redissolved in
chaotropic agent, iB subjected to gel filtration to
obtain fractions of molecular weight below about 30,000
daltons, thus resulting in a major enhancement of
purity. Gel sizing is done using standard technique6,
preferably on a Sephacryl column at room (10C-25C~
temperature.
The low molecular weight fraction is then
subjected to ion exchange chromatography using CMC at
approximately pH 4.5-5.2, preferably about 4.8, in the
presence of a nonionic chaotropic agent such as 6 M urea.
Other cation exchangers may be used, including those
derived from polyaerylamide and cross-linked dextran;
however cellulosic cation exchanger6 are preferred. Of
course, as in any ion exchange procedure, the solution
must be freed of competing ions before application to
the column. The factor is ad~orbed on the column and i6
eluted in an increasing salt concentration gradient in
the range of about 10 mM to about 150 mM.
The 10 mM-150 mM NaCl fraction from the cation
exchange column may be subjected ~o ~P-HPLC or
~L~6~35
nondenaturing gel electeophore6is for further
pucification.
The presence of the factor in the 10 mM-150 mM
NaCl fraction is confirmed using an in vivo
bone-induction assay described in detail below.
Example
The following example i8 intended to illustrate
the process for purification as applied to a particular
sample. It is not intended to limit the invention.
. Preparation of Demineralized Bone
Fresh bovine metatarsal bone was obtained fresh
from the slaughterhouse and transported on dry ice. The
bones were cleaned of marrow and non-bone tissues,
broken in fragment6 smaller than 1 cm diameter, and
pulverized in a mill at 4C. The pulverized bone was
washed twice with 9.4 liters of double distilled water
per kg of bone for about 15 min each, and than washed
overnight in 0.01 N HCl at 4C. Washed bone was
defatted u~ing 3 X 3 volumes ethanol, followed by 3 X 3
volumes diethylether, each washed for 20 min, and all at
room temperature. The resulting defatted bone powder
was then demineralized in 0.5 N HCl (25 l/kg defatted
bone) at 4C. The acid was decanted and the resulting
DMB washed until the wash pH was greater than 4, and the
DMB dried on a suction filter.
B. Extraction of Noncollaqenous Proteins
The DMB as prepared in 1~A was extracted with
3.3 1 o~ 4 M guanidine-HCl, 10 mM ethylenediamine-
tetraacet:ic acid (EDTA), pH 6.B, 1 mM PMSF, 10 ~M NEM
per kg for 16 hr, the 6uspension suction filtered and
the nonsoluble material extracted again for 4 br. The
~LZ~6~3S
soluble fractions were combined and concent~a~ed at
lea~t 5-fold by ultrafiltr,ation using an ~micon
ultrafiltration (lOK) unit, and the concentrate dialyzed
against 6 change~ of 35 volumes cold deionized water
over a period of 4 days, and then lyophilized. All of
ehe procedure~ of thi6 par,agraph were conducted at 4C
except the lyophilization which was conducted under
standard lyophilization conditions.
lo C. Gel Filtration
The ex~ract from 9~B, redi6solved in 4 M
guanidine-HCl, wa~ fractionated on a Sephacryl S-200
column equilibrated in 4 M guanidine-HCl, 0.02% sodium
azide, 10 mM EDTA, pH 6.8. Fractions were assayed by
their absorbance at ~80 nm and the fractions were
combined as shown in Figure 1. Fraction F2 of Figure 1,
constituting a low molecular weight (LMW, 10,000-30,000
daltons) protein fraction po6sessing the greatest
activity was dialyzed against 6 changes of 180 volumes
of deionized water and lyophilized. ~11 operations
except lyophilization and dialysis (4C) were conducted
at room temperature.
D. Ion Exchanqe_Chromato~raPhV
Fraction F2 from ~C was di6solved in 6 M urea,
10 mM NaCl, 1 mM NEM, 50 mM ~odium acetate, pH 4.8 and
centrifuged at 10,000 rpm for 5 min. The supernatant
wa6 fractionated on a CM52 (a commercially available
CMC) column equilibra~ed in the 6ame buffer. Bound
proteins were eluted from the column using a 10 mM to
400 mM NaCl gradient in ~he same buffer, and a total
volume of 350 ml at a flow rate of 27 mlJhr. Three
major fraction6, designa~ed CM-l, CM-2 and CM-3, were
collected as 6hown in Figure 2. Each frac~ion wa~
6~3~i
dialyzed against 6 changes of 110 volume~ of deionized
water for ~ days and lyophilized. All of the foregoing
operation6 were conducted at room temperature except
dialy~is (4OC).
E. RP-HPLC
The lyophilized fraction6 CM-2 and CM-3 from 1~D
were each di6solved in 0.1% trifluoLoacetic acid (TFA)
and aliquots of the ~olution loaded onto a Vydac C18
RP-HPLC column (4.6 mm ID X 25 cm) and washed with 0.1%
TFA for 5 min at 1 ml~min. The eluting ~olvent was a
0%-60% acetonitrile gradient in 0.1% TF~ at a rate of
2%/min. Two peaks were obtained--peak A at about 2~.5
min and peak B at about 31.3 min.
F. ~ssay for Chondroqenic ~ctivi~
The pre6ence of the de~ired proteins in the
fractions during purification was confirmed using an in
vitro assay for the production of proteoylycans (PG),
the identity of which was confirmed by enzyme-linked
immunosorbent assay (ELISA). The assay is an agarose
gel culture model u~ing leg muscle cell6 isolated from
rat fetuses. It a6ses~es the ability of the ~amples to
induce the production of cartilage ~pecific PG~. The
correlation between ln vitro cartilage induction and in
vivo bone formation has been ~hown by Seyedin, S., et
al. J Cell Biol (1983) 97:1950-1953.
The cell culture wa6 prepared by removing
muscle tissue a~eptically from the upper limbs of
nineteen-day-old Sprague Dawley rat fetu6es, mincing the
ti~sue, and culturing it in Eagle's Minimum Es6ential
Medium (MEM) with 10% fetal bovine serum (FBS) and 50
units ~enicillin, 50 g streptomycin per ml. Cellular
outgrowth usually reached conflsency within one week,
3S
-- 10 --
whereupon cells were trypsini~ed, split 1:2 and used for
experimentation within the firsl: three passages.
The cells were placed in agarose gel cultures either
with control medium or with samples to be tested. The
procedure was basically that of Benya, et al, Cell (1982)
30:215. Briefly, the cell rnonolayers were harvested by
trypsinization, counted on a hemocytometer, and resuspended
at two times the final cell concentration in the medium with
or without the protein fraction to be tested. The control
medium was either Hamæ F12, Dulbecco's Minimum Essential
Medium (DMEM) or CMRL 1066 (Gibco) each containing 10% FBS
and antibiotics. The test protein fractions in 1.01 N HCl
were diluted directly to the desired concentration of test
protein diluted with an equal volume with 1% low melting
agarose (Bio-Rad*, #162-0017) in F-12, and 0.2 ml of the
dilution was plated on 17 mm wells coated with 0.15 ml of 1%
high melting (Bio-Rad, #162-0100) agarose. The resulting
cultures were incubated at 37C for 5 min, chilled at 4C for
10 min, and then overlayed with 1 ml of the corresponding
medium (control or test protain~. The cells were then
cultured in a humidified atmosphere of 5% C02, 95% air and
fed every 3-4 days thereafter by a complete change with
control medium. Aft&r 7 days the cultures were frozen and
stored at -80C before assay.
The cultures were assayed by thawing at 4C,
homogenizing in 4 M guanidine-HCl with 50 mM Na acetate, 13
mM EDTA, 6 mM NEM, and 3 mM PMSF at pH 5.8, and extracting by
shaking overnight at 4C. The supernatant fraction from
centrifugation at 25,000 X g for 40 min at 4C was dialyzed
overnight at 4 C against 50 volumes 0.2 M NaCl, 50 mM Tris,
pH 7.4~ The supernatant was assayed
(*) Trademark
3~
for proteoglycans by ELISA a6 described by Renard, et
al, Anal Biochem (1~80) 104:205, and in U.S. 4,434,094.
Briefly, for the E~LISA, antiserum to cartilage
PGs was raised in ~abbits using 6tandard techniques
which 6howed no cross-reactivity with hyaluronic acid or
PGs extracted from rat bone. Purified proteoglycan
(Seyedin, S., e-t al, supra) from Swarm rat
chondrosarcoma tis6ue wa~ used as standard antigen. ~he
dialyzed samples were diluted 1:1 (v/v) in
phosphate-buffered saline (PBS) with 0.05% Tween 20, 1
mg/ml bovine serum albumin (BSA), pH 7.2 for assay.
horseradish ~eroxidase conjugated goat anti-rabbit IgG
(Tago) was the second antibody with o-phenylenediamine
as substrate.
The results of the ELISA of the three CM-bound
~rac~ions from 1~D (designated CMC-B-l, CMC-B-2, and
CMC-B-3) and the protein of peak ~ (designated CIF-~)
from 1~E are shown in Figures 3(a) and 3(b).
G. Reconstitution Procedure
For reconstitution, the proteins prepared in
the above described manner were combined with a 9:1
weight ratio mixture of bone collagen powder (BCP,
lyophilized 601idg from 91C) and collagen in solution
(CIS, available commercially from Collagen Corporation,
-~ Palo ~lto, California under the trademark ZYGE~a)
containing 10~ native 601uble bovine skin collagen by
weight in ratios according to their in vitro
chondrogenic activitieg. The procedure is depicted
gchematically in Figure 4 and details of the
compo6itions of the reconstituted (R) materials are
reported in ~he Table 6hown in Figure 5. According to
thi~ method all the reconstituted samples contained
approximately an equal number o~ unit~ of chondrogenic
3S
activity (about 1000 units/100 mg ~-DBP). Only for
CIF-A the do6age was doubled and CMC-B-l wa6
recon6tituted in two diffeeent do6ages. The protein
content of CMC-B-l was determined by Biuret assay and
the one of all the other 6amples was measured by
integration of the E3PLC peaks compared to the peak aLea
of a known bovine 6erum albumin ~tandard at 220 nm. In
case of pure CIF-~ and CIF-B the HPLC feactions were
directly added to the CIS solution before mixing with
BCP. ~lso, a control sample consisting of the BCP/CIS
carrier was prepared under the same conditions.
H. BioassaY SYstem
The osteoinductive abilities of samples were
assayed by their ability to induce endochond~al bone
formation in the subcutaneous tissue of young male
Sprague~Dawley rats. The samples were wetted with two
volumes of sterile double distilled water (v/w),
thoroughly mixed, packed in a 1 cc syringe, cut and
20 weighed. ~11 the samples were implanted on the ventral
thoracic region, one on each side of the animal.
Explant6 were removed afte~ 14 and Z8 days and evaluated
biochemically and histologically.
I- Histolo~y Studies
Explants which had been removed after 14 and 28
days were subjected to histological as6e66ment by fixing
in 10% neutral formalin for 26 hr, and then proce6sing
~or paraffin embedding. Four-six micron 6ections were
taken from the imbedded tissue6 and were 6ubseguently
stained with either hematoxylin-eo~in (general
cytology), with 6afronin-O (proteoglycans) and Gomori
trichrome (collagen).
643S
-13-
J. Biochemical Assays
The 14 day explants weee 6plit in half, the wet
weight determined and frozen at -B0C till processed.
The samples were first extcacted and assayed for
alkaline phosphatase activity and subsequently extracted
and as~ayed for cartilage-specific proteoglycans. The
right side 28 day explants were extracted and as~ayed
first for alkaline phosphatase and then for calcium.
The extraction and assay procedure6 are de~cribed below.
J.l. ProteoqlYcan l~ssay
Cartilage proteoglycan was assayed by an ELISA
technique. The explants were weighed immediately after
removal and fLozen at -70C until extraction. For t~e
extraction, the explants were cut into slices, and
homogenized in ice cold extraction buffer in a Tekmar
Tissuemizer for two 30 sec burst6 at maximum setting.
The extraction buffer was 6 ~ guanidine hydrochloride,
75 mM sodium acetate or 4 M guanidine hydrochloride, 50
mM acetate both containing 20 mM EDTA, 1 mM PMSF and 10
mM NEM at pH 5.8. Bufer was used in a 10:1 volume to
~he weight of the explant extracted, and the samples
were incubated overnight ~20 hr) a~ 4C. The samples
were then centrifuged at 12,000 rpm for 1 hr at 4~C, and
the ~upernatant~ dialyzed overnight at 4C again6t 50
volumes of 50 mM Tris, 200 mM NaCl, pH 7.~. The
dialyzate was subjected to ELISA performed as described
by Renar~, et al, ~rch Biochem Bioph~s (19aO) 207:399
and by Seyedin, S., et al, J Cell ~iol (1983~ g7:1950
u8ing poly~tyrene microplates (Flow Laboratories,
McClean, Virginia). Tha antisera and the ~roteoglycan
standard were prepared from Swarm rat chondro6arcoma
ti~sue aq~ de~cribed by Seyedin, S., et al, (supra).
Horseradish peroxida6e conjugated goat anti-rabbi~ IgG
~266~3~S
- 14 -
was used as the second antibody, samples were assayed, in
different solutions in PBS, 0.05% Tween* 20, 1 mg/ml BSA and
quantitated by use of the inhibition 2LISA described by
Shuures, et al, Clin Che_ (1977) 81:1.
J.2. Extractable Calcium
The formation of bone was also assessed by
determination of calcium. The explants were cut in small
pieces and suspended in 1:10 (m/v) of 0.5 N HCl to dissolve
the ions. The samples were incubated for another 5 days at
room temperature and centrifuged at 12,000 rpm for 40 min.
The calcium concentration of the supernatant was determined
by atomic adsorption (Trace Analysis Laboratory, Hayward,
California).
J.3. Analysis for Alkaline Phosphatase
To determine alkaline phosphatase (AP), the explants
were cut in small pieces and homogenized in 10 volumes tl/10)
of ice cold 1.5 M NaCl, 3 mM NaHC03, pH 7.5. The homogenized
samples were then centrifuged at 12,000 rpm for 50 min at
4C, and an aliquot of the supernatant diluted 1:10 in cold
distilled water. The method of Huggins, et al, J Exp Med
(1961) 114:761 was used to assess alkaline phosphatase using
polystyrene plates.
K. Results of Histolo~y Studies and Biochemical
Assays
Partial results are summarized in the table below.
(*) Trademark
~ 26~435
-15-
SUMMARY: HISTOLOGY AND ~LKALINE PHOSPHATASE ACTIVITY
BCP/CIS COMBINED WITH CMC-FRACTIONS
Group Cartilage Bone AP Units/
~ w.Ti~sue (Explant6)
At Day 14 Post Implantation
R-CMC-B 5+ 3~ 10.1
(149:~)
R-CMC-B-l 2+ 4+ 11.5
(62:1)
BCP/CIS o o o oO
(9:1)
~t DaY 28 Post ImPlan-tation
R-CMC-B 3+ 4~ 11.0
(1~8:1)
R-CMC-B-l + 5~ 13.2
(62:1)
BCP/CIS O O
(Carrier alone)
The above data show that CMC-B-l proteins
enhance osteoinduction relative to CMC-bound (the ~otal
bound fraction) as reflected by a higher ra~e, quantity,
and quality of bone formation. These studies indicate
that endochondral bone formation is affected by the
purity of the bone-inducing material and the
di6tribution of proteins therein. The~e ~tudies further
suggest that the two proteins identified in European
Patent ~pplication 85.304B48.6 may play role6 in the
differentia~ion of cells involved in bone formation and
affect rate and relative amounts of cartilage and bone
formation.
~s indicated, histological and biochemical da~a
of the 1~ and ZB day~ explanted materials demonstra~ed
that reconstituted total CMC-bound (to~al proteins bound
by CMC) and CMC-~-l induced cartilage and bone formation
in all implant~. At 14 days cartilage formation wa~
~Z616~3~
-16-
very high with the R-CMC-bound implantg and uniformly
distributed over the whole implant. Some ne~ bone wa~
formed peripherally. In contrast, R-CMC-B-l showed only
little cartilage and already lots of bone at 14 days.
At 28 days R-CMC-bound expLants still contained
cartilage. (The amount of cartilage and bone appeared
to be about the same.) R-CMC-B-l meanwhile, contained
only traces of cartilage and well-developed bone with
fatty marrow cavities. ~11 of the latter explants
appeared to be larger than usually found~ Hi6tological
ob6ervations were confirmed by biochemical data. Both
materials 6howed high levels of alkaline phosphata6e
activity. The calcium content at 28 days was very high
in both materials (approximately ~2 mg Ca/g wet tissue
for R-CMC-bound and approximately 39 mg Catg wet tis6ue
for R-CMC-B-l).
The claimed 06teogenic material may be u6ed as
the active ingred~ient of 06teogenic implant compo6itions
for repairing, replacing, or augmenting bone tis6ue in
living mammal6, including man. Ogteogenically effective
amounts of the material will normally be formulated with
pharmacologically and physiologically acceptable solid
carriers such a~ BCP for implantation. The weight ratio
of active protein to carrier will tyeically be in the
range of 1:50 to 1:1000. The implants may be placed at
a predetermined 6ite in the patient by conventional
6urgical teshnique6.
The claimed factor may al~o be useful for
treating bone deficiencie6, such as osteoporosi6 and
06teopetrosis, systemically. FOL ~uch trea~ment the
proteins will be formulated in therapeutically effective
amounts with injectable carriers and administered
parenterally to the patient.
