CA1266435A - Partially purified bone-inducing factor - Google Patents

Abstract

PARTIALLY PURIFIED BONE-INDUCING FACTOR

Abstract A partially purified protainaceous bone-inducing factor of 10,000 to 30,000 daltons is described. It is derived from demineralized bovine bone by extraction with a chaotropic agent, gel filtration, cation exchange chromatography using carboxymethyl cellulose at pH 4.8 and gradient elution with NaCl at 10 mM to about 150 mM.

Description

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;

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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, 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 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).

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

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 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~

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.

Claims (6)

WHAT IS CLAIMED IS:

1. A process for preparing a partially purified bone-inducing factor which comprises:
(a) treating demineralized bone with a chaotropic extractant that solubilized nonfibrous proteins;
(b) subjecting the extract from step (a) to gel filtration to recover a fraction containing proteins of molecular weight 10,000-30,000;
(c) adsorbing the fraction from step (b) onto a carboxymethyl cellulose cation exchanger at approximately pH4.5-5.5; and (d) eluting an osteogenically active fraction from the cation exchanger with a sodium chloride gradient of about mM to about 150 mM in the presence of a nonionic chaotropic agent.

2. The method of claim 1 wherein the extractant is guanidine hydrochloride and the pH is about 4.8.

3. A partially purified bone-inducing factor prepared by the process of claim 1.

4. A partially purified bone-inducing factor prepared by the process of claim 2.

5. An osteogenic implant material comprising:
(a) an osteogenically effective amount of a factor of claim 1 reconstituted with (b) a substantially nonimmunogenic carrier.

6. An ostoegenic implant material comprising:
(a) an osteogenically effective amount of a factor of claim 2 reconstituted with (b) a substantially nonimmunogenic carrier.