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PCT 



WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




INTERNATIONAL APPLICATION PUBLISHED UNPER THE PATENT COOPERATION TREATY (PCT) 



(51) International Patent Classification 6 : 
A61L 27/00, A61F 2*28 



Al 



(11) International Publication Number: WO 96/39203 

(43) International Publication Date: 12 December 1996 (12.12.96) 



(21) International Application Number: PCT/US96/09749 

(22) International Filing Date: 6 June 1996 (06.06.96) 



(30) Priority Data: 
08/469,982 



6 June 1995 (06.06.95) 



US 



(71) Applicant: BIOCGLL LABORATORIES, INC. [US/US]; Suite 

600, 562-lst Avenue South, Seattle, WA 98104 (US). 

(72) Inventor: JEFFERIES, Steven, R.; 3692 Wingficld Drive, 

York, PA 17402 (US). 

(74) Agents: MOROZ, Eugene et al.; Morgan & Finncgan, L.L.P., 
345 Park Avenue, New York, NY 10154 (US). 



(81) Designated States: AL, AM, AT, AU, AZ, BB, BG, BR, BY, 
CA, CH, CN, CZ, DE t DK, EE, ES, Fl, GB, GE, HU, IL, 
IS, IP, KE, KG, KP, KR. KZ. LK, LR, LS, LT, LU, LV, 
MD, MG, MK, MN, MW, MX, NO, NZ, PL. PT, RO, RU, 
SD, SE, SG ( SI, SK, TJ, TM, TR, TT, UA, UG, UZ, VN, 
ARIPO patent (KE, LS, MW, SD, SZ, UG). Eurasian patent 
(AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European patent 
(AT, BE, CH, DE, DK, ES, FI, FR, GB, GR. IE, IT, LU, 
MC, NL, PT, SE), OAPI patent (BF, BJ, CF, CG, CI, CM, 
GA, GN, ML, MR, NE, SN, TD, TG). 



Published 

With international search report. 

Before the expiration of the time limit for amending the 
claims and to be republished in the event of the receipt of 
amendments. 



(54) Title: MODIFIED OSTEOGENIC MATERIALS 
(57) Abstract 

A process and product comprising collagen and demineralized bone particles. The product may con ^* * ^ 
weight in^ic materials. The product may be densified by compression. Additional osteogenic factors mitogens ^ 
rr^Vrated therein. Inorganic materials may be bound to the organic matrix ™ P™**^ 

binding prote^peptide or amino acid. The materials also display long lasting drug release charactensucs. ^ejubjert of this inventus n ; a 
process and result composition which increases the rate and predictability of osteoinduction by d f™ c ^^^^ 

Evention relates to compositions of demineralized bone and calcium or omer mineral sal* > which ^^^ff^^^ 
This invention further relates to osteogenic composiUons comprising between about 60 % to 90 % demineralized bone by weight and to 
compositions comprising a carrier and alkaline phosphatase capable of inducing bone-hke structures. 



FOR THE PURPOSES OF INFORMATION ONLY 



Codes used to identify States party to the PCT on the front pages of pamphlets publishing international 
applications under the PCT. 



AM 


Armenia 


GB 


United Kingdom 


AT 


Austria 


GE 


Georgia 


AU 


Australia 


GN 


Guinea 


BB 


Barbados 


GR 


Greece 


BE 


Belgium 


HU 


Hungary 


BF 


Burkina Faso 


IE 


Ireland 


BG 


Bulgaria 


IT 


Italy 


BJ 
BR 


Benin 


JP 


Japan 


Brazil 


KE 


Kenya 


BY 


Belarus 


KG 


Kyrgystan 


CA 


Canada 


KP 


Democratic People's Republic 


CF 


Central African Republic 




of Korea 


CG 


Congo 


KR 


Republic of Korea 


CH 


Switzerland 


KZ 


Kazakhstan 


a 


Cote d'lvoire 


U 


Liechtenstein 


CM 


Cameroon 


LK 


Sri Lanka 


CN 


China 


LR 


Liberia 


CS 


Czechoslovakia 


LT 


Lithuania 


CZ 


Czech Republic 


LU 


Luxembourg 


DE 


Germany 


LV 


Latvia 


DK 


Denmark 


MC 


Monaco 


EE 


Estonia 


MD 


Republic of Moldova 


E5 


Spain 


MG 


Madagascar 


FI 


Finland 


ML 


Mali 


FR 


France 


MN 


Mongolia 


GA 


Gabon 


MR 


Mauritania 



MW 


Malawi 


MX 


Mexico 


NE 


Niger 


NL 


Netherlands 


NO 


Norway 


NZ 


New Zealand 


PL 


Poland 


PT 


Portugal 


RO 


Romania 


RU 


Russian Federation 


SD 


Sudan 


SE 


Sweden 


SG 


Singapore 


SI 


Slovenia 


SK 


Slovakia 


SN 


Senegal 


sz 


Swaziland 


TD 


Chad 


TG 


Togo 


TJ 


Tajikistan 


TT 


Trinidad and Tobago 


UA 


Ukraine 


UG 


Uganda 


US 


United States of America 


uz 


Uzbekistan 


VN 


Viet Nam 



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- 1 - 

TITLE OF THE INVENTION 
MODIFIED OSTEOGENIC MATERIALS 



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PRIOR APPLICATIONS 

This application is a continuation-in-part of 
U.S. patent application Serial Number 08/422,745 filed 
April 14, 1995 which is a continuation of application 
serial number 08/057,951 filed January 29, 1993, which is 
a continuation of application serial number 07/892,646 
filed June 2, 1992, which is a continuation of application 
serial number 07/718,914 filed of June 24, 1991, which is 
a continuation of application serial number 07/119,916 
filed November 13, 1987 which is a continuation-in-part of 
application serial number 80,145 filed July 30, 1987 which 
is a continuation of application serial number 844,886 
filed March 27, 1986. 

FIELD OF THE INVENTION 
The present invention relates to bone repair 
materials with improved cohesive and physical strength for 
use in stress -bearing defects or where the ability to 
produce and maintain the specific shape of an implant is 
important. The principle of creating a stable interface 
and conjugate between a protein-based particle and an 
organic matrix is also applicable to drug delivery 
materials and devices. This invention also relates to 
osteogenic bone repair compositions having enhanced 
osteogenic potential. In particular, to compositions of 
demineralized bone arid soluble calcium or mineral salts 
and to methods for preparing these bone repair 
compositions having enhanced osteogenic potential and to 
therapeutic uses for these compositions. 

narKftROUND ART 
The repair of osseous defects involves either 
non- resorbable or resorbable prosthetic structures. The 
resorbable structures or materials either support the 



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ingrowth of adjacent bone and soft tissue or actively 
induce the formation of new bone. This active formation 
of new bone, termed osteoinduction, occurs only in the 
presence of demineralized bone matrix or in the presence 
of protein extracts from such matrix, or a combination of 
5 both materials. Particles or powders produced from 
demineralized bone matrix possess greater osteogenic 
potential per unit weight due to their increased surface 
area, than blocks or whole segments of demineralized bone. 

Other methods of repairing damaged or missing 
10 osseous tissue or bone have also been explored. 

Replacement or support with nonresorbable materials, such 
as biocompatible metals, ceramics, or composite 
metal -ceramic materials, offers one method of clinical 
treatment. Some of these materials, such as metal grade 
15 titanium, can promote osteocoinduction at their surface, 
thus leading to a stable, continuous interface with bone. 
Caffessee et al. Journal o f Periodontologv. Feb. 1987 
utilizing a "window" implantation technique, established 
that nonabsorbable ceramics, such as hydroxyapatite, fail 
20 to stimulate tissue, even when placed in osseous defects. 

Resorbable ceramics, such as tricalcium phosphate, display 
better conduction of mineralized tissue into the resorbing 
graft material when placed in osseous defects. Unlike 
demineralized bone matrix, tricalcium phosphate or 
25 hydroxyapatite fail to stimulate induction of new bone 
when placed in non- osseous tissue. The addition of 
tricalcium phosphate or hydroxyapatite to demineralized 
bone matrix or to the extracted bone- inducing proteins 
actually inhibits the osteogenic potential of these 
30 established osteoinductive compositions (see Yamazaki et 
al- Experimental Study On the Osteoinduction Ability of 
Calcium Phosphate Biomaterials with added Bone 
Morphogenetic Protein Transactions of the Society For 
FHnmaterials pg 111, 1986.) 



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10 



Aside from the documented inability of 
hydroxyapatite and tricalcium phosphate ceramic materials 
to independently induce osteogenesis, recent clinical 
findings indicate that osteointegration of inorganic 
particles is highly dependent on the ability of those 
particles to remain fixed in a definite position, 
preferably near a bony interface. Hence, the immobility 
of the parties is a prerequisite for involvement with new 
bone formation (See Donath, et al., A Histologic 
Evaluation of a Mandibular Cross Section One Year After 
Augmentation with Hydroxyapatite Particles Oral Surgery, 
Oral Medirine. Ora l Pathology vol 63 No. 6 pp. 651-655, 
1987. 

Nevertheless, numerous compositions have been 
derived to create clinically useful bone replacement 

15 materials. £ru2 U.S. Patent No. 3,767,437 describes 

artificial ivory or bone- like structures which are formed 
from a complex partial salt of collagen with a metal 
hydroxide and an ionizable acid, such as phosphoric acid. 
With regard to the metal hydroxide, this composition 

20 stresses the use of a polyvalent metal cation in the metal 
hydroxide, such as calcium hydroxide. Calcium phosphate 
may be added to the complex collagen salt. Cruz also 
recites the. addition of fibers and ions to increase 
hardness and structural strength, but does not document or 

25 make claims with regard to these specific improvements. 
Cruz does not mention or claim these compositions to be 
osteoinductive or osteoconductive, nor does he mention 

their behavior in- vivo. 

Thi p! p . et al . . U.S. Patent No. 4,172,128, 
30 recites a process of degrading and regenerating bone and 
tooth material and products. This process involves first 
demineralizing bone or dentin, converting the 
demineralized material into a mucopolysaccharide -free 
colloidal solution by extraction with sodium hydroxide 
adding to the resultant solution a physiologically inert 



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foreign mucopolysaccharide, gelling the solution, and then 
remineralizing the resulting gel. Thiele et al. indicate 
this material to be biocompatible and totally resorbable, 
thus replaced by body tissue as determined by histologic 
analysis the gel material produced by this process is 
5 reported to completely replace destroyed bone sections 
created in experimental animals. The patentees do not 
indicate any ability by the material to induce new bone. 
The ultimate fate of these materials in-vivo-, or their 
ability to stimulate the formation of new bone in non- 
10 osseous implant sites is not described. The patentees do 
not describe or quantify the strength properties of these 
material. Nevertheless, since they are described as gels, 
one can assume their strength to be low. 

Urist In U.S. Patent No. 4,294,753, describes a 
15 process of extracting and solubilizing a Bone 

Morphogenetic Protein (BMP) . This is a glycoprotein 
complex which induces the formation of endochondral bone 
in osseous and non- osseous sites. This partially purified 
glycoprotein, which is derived from demineralized bone 
20 matrix by extraction, is lyophilized in the form of a 

powder. Urist describes the actual delivery of BMP in in- 
vivo testing via direct implantation of the powder, 
implantation of the powder contained within a diffusion 
chamber, or coprecipitation of the BMP with calcium 
25 phosphate. While Urist describes the induction of new 

bone after the implantation of one of these forms of BMP 
in either osseous or non- osseous sites, Urist fails to 
address the intrinsic physical strength properties of any 
of these delivery forms. Lyophilized powders and calcium 
30 phosphate precipitates, however, possess little if any, 

physical strength. Furthermore, more recent investigators 
(see aforementioned Yamazasaki, et al.) indicate that 
calcium phosphate ceramics, such as tricalcium phosphate 
and hydroxyapatite, when present in high concentrations 



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° relative to the BMP present, may actually inhibit the 
osteogenic action of the BMP. 

Jeffries in U.S. Patent No. 4,394,370 and 
4,472,840 describes bone graft materials composed of 
collagen and demineralized bone matrix, collagen and 
5 extracted Bone Morphogenetic Proteins (BMP). Also 

described is a combination of collagen, demineralized bone 
matrix, plus extracted bone morphogenetic proteins. 
Jeffries describes an anhydrous lyophilized sponge 
conjugate made from these compositions which when 
10 implanted in osseous and non- osseous sites, is able to 

induce the formation of new bone. The physical strength 
of these sponges is not specific in the disclosure, 
however, reports of the compressive strength of other 
collagen sponges indicates these materials to be very weak 
15 and easily compressible (much less than l kilogram load 
needed to affect significant physical strain in 
compression or tension) . 

Smestad in U.S. Patent No. 4,430,760 assigned to 
Collagen Corporation, describes a nonstress- bearing 
20 implantable bone prosthesis consisting of demineralized 

bone or dentin placed within a collagen tube or container. 
As the patentee indicates, this bone prosthesis can not be 
used in stress -bearing locations clinically. 

n ^warki et al. . in U.S. Patent No. 4,440,750 
25 apparently assigned to Collagen Corporation and Harvard 
University describe plastic dispersions of aqueous 
collagen mixed with demineralized bone particles for use 
in inducing bone in osseous defects. This graft material, 
as described exists in a gel state and possess little 
30 physical strength of its own. It use, therefore, must be 
restricted to defects which can maintain sufficient form 
and strength throughout the healing process. Furthermore, 
with time, the demineralized bone particle suspended 
within the aqueous collagen sol -gel begin to settle under 



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gravitational forces, thus producing an nonhomogeneous or 
stratified graft material. 

fig Y pdin - et a1 - - in u - s - patent No - 4,434,094, 
describes the purification of a protein factor, which is 
claimed to be different than Urist's BMP molecule, 

5 responsible for the induction of chondrogenic activity. 

Bell , in U.S. Patent 4,485,097, assigned to 
Massachusetts Institutes of Technology, describes a bone 
equivalent, useful in the fabrication of prostheses, which 
is composed from a hydrated collagen lattice contracted by 

10 fibroblast cells and containing demineralized bone powder. 
As this prosthetic structure is also a hydrated collagen 
gel, it has little strength of its own. The patentee 
mentions the use of synthetic meshes to give support to 
the hydrated collagen lattices to allow handling. 

15 Nevertheless, there is no indication of the clinical use 
of the material or measurement of its total physical 
strength. 

Rp-ifi. et al. . in U.S. Patent No. 4,623,553, 
describes a method for producing a bone substitute 

20 material consisting of collagen and hydroxyapatite and 

partially crosslinked with a suitable crosslinking agent, 
such as glutaraldehyde or formaldehyde. The order of 
addition of these agents is such that the crosslinking 
agent is added to the aqueous collagen dispersion prior to 

25 the addition of the hydroxyapatite or calcium phosphate 
particulate material. The resultant dispersion is mixed 
and lyophilized. The patent lacks any well known 
components which are known osteogenic inducers, such as 
demineralized bone matrix or extracted bone proteins. 

30 ra plan. et al. . in U.S. Patent No. 4,620,327, 

describes a method for treating implants such as a 
biodegradable masses, xenogeneic bony implants, 
allografts, and prosthetic devices with soluble bone 
protein to enhance or stimulate new cartilage or bone 

35 formation. These structures may then be crosslinked to 



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- 7 - 

immobilize the soluble bone protein or retard its release. 
While the osteogenic activity of these implants are 
described in detail, their physical strength is not 
mentioned. 

The above review of the prior art reveals that 
5 none of the bone prosthetic materials which claim the 
ability to induce new bone formation (osteoinductive 
materials) possess high strength characteristics. 
Furthermore, of those materials which are described with 
enhanced strength, these materials consist solely of a 
10 crosslinked conjugates of collagen and inorganic mineral, 
which lacks the ability to stimulate the induction of new 
bone. 

It is especially relevant that none of the above 
references address the need to bind the dispersed 
15 particulate or inorganic phase to the organic carrier 

matrix (i.e. collagen). As will be described below, the 
treatment of demineralized bone matrix or particles or 
inorganic particles, prior to complexation with an organic 
biopolymer, such as collagen, is extremely important in 
20 determining the physical strength characteristics of the 

bioimplant. Furthermore, the ability to orient protein or 
peptide particles in a stable fashion within inorganic or 
natural polymeric matrixes permits the ability to release 
drugs, bioactive proteins, and bioactive peptides in a 
25 controlled fashion. 

As discussed above a variety of bone graft 
materials are available to repair, replace or regenerate 
bone lost to disease or injury. Bone grafts may be 
allografts, meaning processed biologic bone material 
30 derived from donors of the same species; or alloplastic, 
meaning not derived from biologic materials and composed 
solely of inorganic or synthetic polymeric materials. 
Bone graft materials can also be classified as 
osteoinductive or osteoconductive. Osteoinductive 
35 mater: Is are capable of inducing the formation of new 



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- 8 - 



10 



bone in both hard tissue defects and, uniquely, in defects 
created in non-bony soft tissue sites in either muscle or 
subcutaneous tissue. Osteoconductive biomaterials cannot 
induce the formation of new bone via differentiation of 
undifferentiated cell types, but do provide a scaffolding 
promote the migration of viable bone tissue from the 
margins of the bony defect along the contacting surfaces 
of the graft material. Because osteoinduction can produce 
new bone even without any available viable bone adjacent 
to he graft material, osteoinductive grafts may be 
preferred to osteoconductive materials . Examples of 
osteoinductive grafts. materials include demineralized bone 
powder and demineralized bone strips or plugs of cortical 
or cancellous bone. 

While a wide variety of osteoinductive 
15 compositions have been used in bone repair and 

regeneration there is always a need in the art for 
improvements or enhancements of existing technologies 
which would accelerate and enhance bone repair and 
regeneration allowing for faster recovery and enhanced 
healing for the patient receiving the osteogenic implants. 

fiT T^RY OF T F R INVENTION 

Currently available or described compositions 
which contain demineralized bone matrix particles or 
conjugates of inorganic particles plus reconstituted 
structural or matrix proteins exhibit poor physical 
stability or physical strength when subjected to load of 
any magnitude. Furthermore, due to the poor structural 
integrity of these materials, further processing into 
alternative shapes or sizes for actual clinical use to 
30 induce new bone formation in osseous defects is limited. 

One of the major objects of this invention is to describe 
a method of producing an osteogenic, biocompatible, 
composite which possesses unique strength properties 
and/or osteogenic properties. While many disclosures in 
the art describe the use of crosslinking agents to enhance 



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25 



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- 9- - 

the physical integrity of protein -based, conjugate, 
osteoinductive materials, this disclosure documents a 
precise method and procedure application which produces 
osteogenic graft materials of exceptional strength and 
physical integrity. 
5 Furthermore, the basic concept described in this 

application, may be adapted to create conjugates of natural 
biopolymers and inorganic bone minerals which display 
exceptional bonds between the inorganic particles and the 
polymeric matrix. The spacial stability of these 
10 particles is critical to their successful use clinically. 

A further object is the creation of protein 
based structures which may release drugs or others agents 
in a controlled and stable fashion. The dimensional and 
physical stability of these conjugate material plays a 
15 significant role in the pharmacologic release properties 

of these materials. Hence, the physical strength and drug 
delivery capabilities are interrelated. 

Two elements are germane to the observed 
properties of these novel compositions. First, the 
20 surface activation and partial cross linking of the 

proteinaceous particles forms a reactive interface such 
that these particles bind in a stable fashion to the 
organic matrix, i.e. reconstituted collagen. This step is 
important with respect to enhanced physical properties. 
25 second, inorganic particles may be bound to and stabilized 
within an organic or protein-based polymer by first 
creating a bound interface of calcium- binding protein or 
peptide to the particle. The modified particle is then 
bound to the matrix proteins via chemical crosslinking or 
30 activation methods. This method, as in the first case, 
significantly enhances the physical properties of these 
conjugates. 

In summary, primary objects of this application 

are: 



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- 10 - 

(1) A method for surface activating and/or 
partially crosslinking protein-based 
or protein coated particles to enhance 
their binding and reactivity to 
organic matrixes, including serum, 

5 plasma, naturally occurring proteins, 

and bone substrates. 

(2) To disclose a method and composition 
which induces bone when implanted in 
an animal or human and has early on 

10 stress -bearing properties not 

described in the prior art. 

(3) To disclose a method and composition 
of binding inorganic particles or 
particles which contain inorganic, 

15 mineral elements to a surrounding 

organic matrix such that a stable, 
stress -bearing conjugate results. The 
inorganic particles in such a 
conjugate are not easily displaced or 

20 dislodged from the matrix, as can be 

the case when the particles are simply 
added to the matrix without 
appropriate surface treatment. 

(4) Applying one of the above methods to 
25 stabilize drug -containing, protein- 
based particles within an organic or 
polymer matrix to effect a delayed or 
controlled release of the drug from 
conjugate material. 

30 (5) A method and composition comprising a 

biocompatible implantable sponge which 
contains a filler component at a 
weight ratio sufficient to enhance the 
resilience of the composite sponge, 

35 \ander both dry and wet conditions, and 



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also to permit maintenance of sponge 
shape, dimensions, and form, even 
under wet conditions . 
This invention also relates to methods of 
surface modification of demineralized bone resulting in 
5 bone graft materials or compositions having enhanced 
osteogenic potential. The osteogenic bone repair 
compositions of this invention having enhanced osteogenic 
potential are used as implants in the treatment of bone or 
periodontal defects. The improved osteogenic compositions 
10 provided herein comprise demineralized bone and at least 
one added calcium or mineral salt. The osteogenic bone 
graft material of this invention, produced by methods 
described herein, exhibit enhanced osteogenic activity 
relative to other bone repair compositions. 
15 it is a general object of this invention to 

provide improved osteogenic bone graft materials 
comprising demineralized bone and at least one calcium or 
mineral salt, wherein said calcium salt or mineral has 
been sorbed onto or into or within the mass of the 
20 demineralized bone or distributed onto the surface or 
within the mass of the demineralized bone. 

It is an object of this invention to provide an 
osteogenic bone graft material having enhanced activity 
comprising demineralized freeze-dried bone powder and at 
25 least one calcium salt or mineral salt wherein said 

calcium or mineral salt has been sorbed onto or into the 
surface of the demineralized bone or distributed onto the 
surface or within the mass of the demineralized bone. 

It is yet another object of this invention to 
30 provide osteogenic bone graft materials having enhanced 

activity comprising demineralized freeze-dried bone powder 
and at least one calcium or mineral salt and at least one 
drug, antibiotic, nutrient, growth factor or blood 
protein. 



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° It is a further object of this invention to 

provide methods of making these improved osteogenic 

compositions. 

It is a further object of this invention to 

provide therapeutic uses for these improved osteogenic 
5 compositions in the repair or replacement of bone or 

periodontal defects. 

It is yet another object of this invention to 

provide osteogenic compositions comprising about 60 

percent to about 95 percent demineralized bone which 
10 exhibit enhanced osteogenic potential and other unique 

properties. 

It is yet another object of this invention to 
provide compositions capable of inducing the formation of 
mineralized bony like structures comprising a carrier and 

15 alkaline phosphatase. 

It is also an object of this invention to 
provide compositions capable of enhancing induction of 
vital new bone in both osseous and non osseous sites 
comprising an osteogenic carrier and alkaline phosphatase. 

20 Further objects and advantages of the present 

invention will become apparent from the description that 
follows . 

DETAILED DESCRIPTION OF THE 
PREFERRED EMBODIMENTS OF TH E INVENTION 

25 When particles which contain protein or amino 

acid components, such as protein microcapsules, finely 
divided particles of reconstituted collagen, demineralized 
bone matrix, or demineralized bone matrix extracted in 
chaotropic agents are partially crossl inked in a low 

30 concentration solution of glutaraldehyde , the surface of 
these particles become highly reactive, thus allowing an 
increased degree of bonding between the particle and an 
organic matrix or polymer, in which the particles may be 
dispersed. These structures, when dehydrated into a solid 

35 mass, display internal cohesive strength properties not 



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13 



° found in simple combinations of the particles dispersed 
within the matrix component . If the glutaraldehyde is 
added directly to the matrix prior to addition of the 
particles and subsequent dehydration, very low levels of 
cohesive strength are developed. This is also true if the 
5 entire dehydrated conjugate matrix is crosslinked. The 
critical element to increasing the strength and internal 
cohesiveness protein-based particle/biopolymer matrix 
conjugate appears to be the partial crosslinking or 
surface activation of only the particles prior to 
10 complexation with the biopolymer organic matrix. 

Alternatively, or additionally, critically controlling the 
actual weight percent of the particle component as weight 
percent of the total conjugate implant can enhance the 
physical properties of sponge configurations as well the 
15 shape and spacing maintaining functions of the implant or 
drug delivery device. 

If bioactive particles, such a demineralized 
bone matrix, or drug containing particles are to be 
complexed, the conditions of surface activation and 
20 partial crosslinking are material. For example, 

crosslinking of demineralized bone particles above .25 
weight percent glutaraldehyde destroys most of the 
osteoinductive capacity of the particles. At higher 
crosslinking levels, the particles will mineralized by the 
25 uptake of calcium phosphate, but will not induce new bone. 
Thus, the use of glutaraldehyde below .25 weight percent 
and, preferably, below .1 weight percent, is a material 
condition in this invention. 

The nature of the matrix effects the ultimate 
30 strength properties of the conjugate biomaterial, which is 
critical in clinical stress-bearing applications. For 
example, reconstituted collagen provides a matrix which 
demonstrates the unique and unexpected strength properties 
of this material. The method in which the collagen is 
35 reconstituted, however, can have a direct effect on the 



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10 



magnitude of the increased cohesive strength. This will 
be illustrated in the Examples which follow. 

Agents other than glutaraldehyde may be used to 
enhance the surface binding of protein-based particles 
within a biocompatible matrix. For example, free and 
available carboxyl groups on the protein particle may be 
converted to amine groups via reaction with a water 
soluble carbodiimide in the presence of a diamine. These 
additional available amine groups can then react with 
glutaraldehyde in the particle crossl inking reaction. 
Alternatively, demineralized bone matrix particles can be 
immersed in solutions of tetracycline which, will enhance 
binding an organic biopolymer matrix. In addition, bone 
particles or partially demineralized bone particles may be 
demineralized in solutions of tetracycline. 
15 Particles with inorganic components may be added 

to these osteogenic stress -bearing compositions, provided 
these particles makeup no more than twenty percent of the 
total weight of the particles. These inorganic component 
particles are bound to the biopolymeric organic matrix via 
20 functional molecules with calcium or hydroxyapatite 
binding functionality. In one embodiment, all the 
particles may be inorganic in nature and bound to the 
matrix in this fashion. The advantage here is enhanced 
strength as well as limiting the loss of particles from 

25 the matrix itself. 

The increased binding between the particle and 
matrix constituents can also be advantageous in drug 
delivery. The method of dispersing a drug, protein, or 
peptide within the particle prior to crosslinking and 
surface activation and permits the use of drug containing 
particles with reduced solubility to act as drug 
reservoirs within a biocompatible matrix. The nature of 
the matrix can regulate the rate of drug release from the 
conjugate material. 



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The matrix biopolymer can be modified in a 
number of ways. For example, the hydrophilic or 
hydrophobic nature of the matrix may be altered by the 
addition of carbohydrates or lipids- The addition of 
acidic phospholipids to the matrix enhances the calcium 
5 binding capacity to the matrix. Additional macromolecules 
may be added to the matrix to achieve a particular 
biologic response. The addition of calcium hydroxide 
whether in a soluble form or as part of a protein-based 
particle, was found to increase the pH of matrix such that 
10 in- vitro bone collagen synthesis was increased in such an 
environment. Heparin may also be added. 

Furthermore, crosslinking agents may be added to 
the matrix or subjected to the entire conjugate to further 
retard the degradation of the matrix and decrease it 
15 solubility. The degree of matrix degradation and its 
inflammatory response can also be controlled by the 
stabilizing affect of alkaline phosphatase. 

Finally, a decided advantage of these 
compositions is their ability to be cast into definite 
20 shapes with good registration of surface detail. Due to 

their structure, there is much greater uniformity in these 
compositions than is found in allogenic tissue. 
Furthermore a significant finding is the ability of these 
conjugates structures to be ground or milled by 
25 conventional means without gross breakdown of the entire 

matrix or the development of severe surface defects. This 
finding is significant since diagnostic techniques now 
allow the accurate three-dimensional representation of 
bony defects with the resultant milling of a graft 
30 material via CAD/CAM technology. There is no other 

processed, truly osteogenic, graft material which can be 
ground to precise specifications for insertion in a bony 
defect. 

The present invention also relates to bone graft 
35 material having enhanced osteogenic potential. The 



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compositions having enhanced osteogenic potential provided 
herein are based on an observation by the inventor that 
the osteoinductive ability of demineralized bone is 
dramatically enhanced by the addition of at least one 
calcium or mineral salt to the demineralized bone. 
5 Furthermore, the composition and method of this disclosure 
greatly increases the speed of bone and mineral formation 
with demineralized bone. 

Material to this invention is a method and 
resultant composition which enhances the mineral content 
10 of demineralized bone by the sorption of a soluble or 
saturated calcium or mineral salt solution, thereby 
producing the unexpected result of enhancing the rate and 
probability of bone formation by osteoinduction as well as 
the quantity of bone induced by a given mass or volume of 
15 demineralized bone matrix. The osteogenic bone graft 

materials provided herein and having enhanced osteogenic 
potential are comprised of demineralized bone and at least 
one soluble calcium or mineral salt. Examples of types of 
demineralized bone that may be used include, but are not 
20 limited to, demineralized bone matrix or partially 

demineralized bone matrix or demineralized or partially 
demineralized freeze-dried bone powder allograft (DFDBA) 
or matrix (DFDBM) . By way of example, the degree of 
demineralization as measured by the weight percent of 
25 calcium remaining in the bone, may range from about 10% 
percent to about 0 weight percent (less than about 0.1 
weight percent) , most preferably, less than about 3 weight 
percent to about 0 weight percent calcium remaining in the 
bone, and most preferred less than about 1 weight percent 
30 to about 0 weight percent calcium remaining in the bone 

after demineralization. Less than about 3 weight percent 
calcium after demineralization is preferred and most 
preferred is less than about 1 weight percent calcium 
remaining after demineralization. A wide range of sizes 
35 and shapes of demineralized bone matrix, ranging from fine 



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powders to coarse powders, to chips, strips, rings, match- 
sticks, wedges, small bone segments a large bone segments, 
may be used in this invention. In a preferred embodiment 
DFDBA is used in the composition. 

The salt may be a calcium or other mineral salt. 
5 By way of example other mineral salts that may be used 
include, but are not limited to, salts such as sodium 
hydroxide, sodium chloride, and magnesium salts, such as 
• magnesium chloride or magnesium hydroxide or other 

biocompatible salts. Examples of calcium salts that may 
10 be used in the methods and compositions described herein 
include, but are not limited to, calcium acetate, calcium 
citrate, calcium chloride, calcium formate, calcium 
glycerophophosphate, calcium lactate, calcium lacerate, 
calcium oleate, calcium oxide, calcium pal is tate, calcium 
15 salicylate, calcium stearate, calcium succinate or calcium 
sulfate. In a preferred embodiment calcium hydroxide is 
used. The salt solutions used in the methods and 
compositions disclosed herein may be at neutral or 
alkaline pH. In a preferred embodiment alkaline pH is 
20 preferred. A soluble or saturated calcium or mineral salt 
solution may be used in the methods described herein. 

By way of example, concentrations of soluble 
salt solution that may be used may range from about 100% 
to about 0.001% of the salt by weight, or may range from 
25 about 10% to about 0.01% of the salt by weight. By way of 
example, for calcium hydroxide, suggested concentrations 
of the solution that may be used may range from about 3% 
to about 0.001% of the salt by weight. 

This invention relates to bone graft 
30 compositions having enhanced osteogenic potential. By way 
of example the weight proportions (weight of the salt 
divided by the pretreatment weight of the demineralized 
bone) of added calcium salt or mineral salt to 
demineralized bone may range from about 0.0001 percent to 
35 about 20 percent or about 0.0010 percent to about 10 



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percent. In a preferred embodiment the composition 
comprises calcium hydroxide to DFDBA at weight proportions 
ranging from about 0.001 percent to about 10 percent. 

This invention also relates to a method for 
producing the osteogenic bone graft compositions having 
5 enhanced osteogenic activity comprising exposing the 

demineralized bone to at least one soluble or saturated 
solution of calcium or other mineral salt, for a time 
sufficient for the ions in the solution to be sorbed into 
or onto the bone matrix or distributed onto the surface or 
10 within the mass of demineralized bone. In a preferred 
embodiment calcium is sorbed onto or into the 
demineralized bone, preferably DFDBA, by sorption of a 
saturated calcium hydroxide solution onto or into the 
structure of the demineralized bone material or 
15 distributed onto the surface or within the mass of the 

demineralized bone. The saturated solution may be at an 
alkaline pH. Alternative methods may be used to prepare 
the compositions of this invention having enhanced 
osteogenic potential. By way of example such methods may 
,20 include, but are not limited to, depositing the calcium or 
mineral salt to the demineralized bone by electrical 
current or plasma discharge. 

Also, intended to be encompassed by this 
invention are functionally equivalent compositions to the 
25 bone repair compositions of this invention having enhanced 
osteogenic potential. 

In an alternative embodiment the demineralized 
bone composition comprising demineralized bone which has 
been exposed to at, least one soluble calcium or other 
30 mineral salt can further comprise demineralized bone that 
has not been exposed to calcium or other mineral salts. 
In addition, if large bone segment or segments are used in 
the methods described herein, the complete bone segment or 
segments used need not be demineralized completely. 
35 Alternatively, only the exposed outer surface of the bone 



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° segment or segments may be demineralized, and then treated 
with calcium or other mineral salt. 

The composition may be dehydrated by 
conventional methods under ambient or elevated temperature 
conditions or may be lyophilized in a commercial range 
5 drier under a wide range of conditions. The composition 
may be in the form of a powder or in the form of 
demineralized bone. strips, chips, segments, assays or 
other sizes and geometries larger and distinct form 
demineralized bone powder. The composition comprising 
10 demineralized bone and added soluble calcium salts or 
mineral salts may be partially activated with a cross 
linking agent by the methods described herein. In yet 
another embodiment of this invention, the calcium or 
mineral salt modified demineralized bone may be admixed 
15 with demineralized bone which has not be modified, or 

alternative, or in addition to, admixed with demineralized 
bone which has been partially activated with a 
crosslinking agent by the methods described herein. The 
weight ratio of each of these various types of powders can 
20 range from about 5 to about 95 percent of the total blend. 
Further, all three types of demineralized bone matrix 
particles can be admixed at a wide variety of ratios to 
create the powder-blend admixture. 

The sorption of a soluble calcium solution or 
25 mineral solution onto and into or within the mass of the 
demineralized bone matrix or distributed onto the surface 
or within the mass of the demineralized bone results in a 
significant increase in both the rate and frequency of 
osteoinduction, when compared to untreated demineralized 
30 bone matrix. The soluble calcium/demineralized bone 

complex also significantly increases the size of induced 
calcified viable bone when compared to equivalent amounts 
of non- calcium enriched demineralized bone as assessed by 
radiograph analysis of mineral formation and histological 
35 analysis of induced bone treated by the compositions and 



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° methods described herein. By way of example, the 

compositions and methods disclosed herein may increase the 
predictability of induction by demineralized bone, to a 
level of about 75%, and more preferably about 90 to 100% 
osteoinduction and mineralization in an animal model. 
5 This invention also relates to osteogenic 

compositions comprised of about 60 weight percent to about 
95 weight percent of demineralized bone, preferably about 
60 weight percent to about 90 weight percent. Examples of 
types of demineralized bone that may be used in these 
10 compositions include, but is not limited to, demineralized 
"bone matrix or partially demineralized bone matrix, 
demineralized or partially demineralized freeze-dried bone 
powder or particles. Examples of materials that may make 
up the remaining about 40 weight percent to about 5 weight 
15 percent or the remaining about 40 weight percent to about 
10 weight percent of the osteogenic composition include, 
but are not limited to, collagen, gelatin, growth factors, 
bone morphogenetic protein (BMPs) , blood proteins such as 
fibrin, albumin or other biocompatible excipients such as 
20 me thy cellulose or hydroxymethyl cellulose. Preferably 
reconstituted collagen is used. The osteogenic 
composition comprising about 60 weight percent to about 95 
weight percent, preferably about 60 weight percent to 
about 90 weight percent demineralized bone described 
25 herein may be fabricated in the form of a dehydrated form 
of a sponge, powder, particles, membrane, fleece or fibers 
by standard methods known to one of skill in the art. 
Sponges can be made by lypholization or controlled 
dehydration under ambient or other control conditions . If 
30 the composition is produced in the form of a sponge, the 
sponge may be ground into particles, powder, or fleece by 
conventional methods. If the composition is in the form 
of a sponge, preferably it is characterized by a density 
of about 0.1 grams/cubic centimeter (cc) or greater than 
35 0.1 grams/cubic centimeters (cc) . By way of example, the 



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range of sponge density may be from about 0.1 grams/cc to 
about 0.5 grams/cc, preferably having a density from 
about 0.11 to about 0.35 grams/cubic centimeter. To 
fabricate sponges with about 90 weight percent or above of 
demineralized bone an acid or alkaline material may be 
5 used to form the remaining balance of the composition. If 
the material to be used is in the acid range, the pH is 
preferably about 5, and most preferably, about 4.5 or 
below. If the material to be used is in the alkaline 
range, the pH is preferably about 9 or above. The 
10 demineralized bone may be combined with the material, when 
the material is in the form of an aqueous solution or a 
dried or lyophilized powder. The lyophilized powder would 
preferably be in the form of an acid or alkaline salt. By 
way of example, collagen or gelatin may be the material 
15 used to form the remaining balance of the compositions. 
Any collagen may be used, preferably mammalian collagen, 
including, but not limited to, human or bovine. 

Yet another embodiment of this invention relates 
to compositions capable of inducing the formation of 
20 mineralized bone- like structures or boney-like structures. 
Such compositions comprise a carrier and alkaline 
phosphatase. Examples of a carrier that may be used 
include, but are not limited to collagen, demineralized 
bone, gelatin, antigen extracted demineralized bone or 
25 demineralized bone matrix extracted with chaotropic agents 
to remove most or all hon- collagen proteins. 

Examples of collagen that may be used include, 
but are not limited to, reconstituted collagen, partially 
demineralized collagen, enzyme extracted collagen or 
30 collagen treated wi.th proteolytic enzymes such as facin or 
pepsin. . The collagen may be at neutral acid or alkaline 
pH. The demineralized bone may be in the form of powder 
or particles. By way of example ranges of alkaline 
phosphatase to carrier that may be are about 10 
35 units/milligram carrier to about 5000 units/milligram 



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carrier, preferably about 100 units /milligram carrier to 
about 1000 units /milligrams carrier. Examples of alkaline 
phosphatase that may be used in these compositions 
includes any mammalian alkaline phosphatase, such as, but 
not limited to, bovine or human. One of skill in the art 

5 will appreciate that the actual weight of alkaline 

phosphatase used in these compositions will vary depending 
on the specific activity of the alkaline phosphatase. 
These compositions may be fabricated in the- form of a 
sponge, powder, particle, fleece, or fiber or membrane by 

10 conventional methodology. Also intended to be encompassed 
by this invention are functionally equivalent compositions 
comprising a carrier and alkaline phosphatase capable of 
inducing bone -like or boney-like structures. 

These materials can be used therapeutically as a 

15 grafting implant in plastic and reconstructive surgery, 
periodontal bone grafting, and in endodontic procedures 
and implanted by standard surgical procedures. The 
osteogenic implants of this invention having enhanced 
osteogenic potential are suitable for both human and 

20 veterinary use. 

All books, articles, or patents referenced 
herein are incorporated by reference. The following 
examples are by way of illustrative aspects of the 
invention but are in no way intended to limit the scope 

25 thereof. 

EXAMPLE ONE 
Ten grams of demineralized bone matrix are 
milled in an A- 10 mill to a uniform particle size ranging 
from 75 to 400 microns. The demineralized bone matrix 

30 particles are sieved to eliminate particles above 400 

microns. Controlling the concentration of glutaraldehyde 
is material to maintaining sufficient osteoinductive 
activity of demineralized bone matrix particles. For 
example, glutaraldehyde crosslinking solutions of as low 

35 as 1.0 to 1.5 weight percent can reduce the residual 



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osteoinductive activity of demineralized bone matrix to 
10% or less. Glutaraldehyde cross linking in aldehyde 
concentrations of .08 or 0.2 weight percent, however, only 
reduce the residual osteoinductive activity of 
demineralized bone matrix by 30 to 35 percent, leaving 
5 from a background osteoinductive activity of from 65 to 70 
percent of uncrosslinked demineralized bone matrix 
particles. Therefore, control of the glutaraldehyde 
concentration used in this procedure is material to 
maintaining the biologic activity of processed 
10 demineralized bone matrix particles. 

The range of glutaraldehyde used to partially 
crosslink and surface activate the demineralized bone 
matrix particle may range from .002 to .25 weight percent 
glutaraldehyde. The preferred range is from .005 to .09 
15 weight percent glutaraldehyde. The partial crosslinking 

of demineralized bone matrix retards the resorption of the 
matrix in a non- inflammatory fashion, enhances the 
attachment of plasma proteins to the surface of 
demineralized bone matrix, and facilitates the attachment 
20 of the demineralized bone matrix to the organic collagen 
matrix of the bony surface of the osseous defect. 

In this example, the demineralized bone 
particles are immersed in a .05 weight percent 
glutaraldehyde aqueous solution buffered with phosphate 
25 buffer to a pH of from 7.0 to 7.6. The glutaraldehyde 
solution is made isotonic by adding NaCl to a final 
concentration of approximately 0.9 weight percent. 
Alternatively, the glutaraldehyde solution may be buffered 
in the acid or the alkaline range. The glutaraldehyde 
30 solution may be unbuffered consisting of only sterile 
distilled deionized water or sterile isotonic saline. 

The demineralized bone matrix <DBM) particles 
are immersed in the solution of .05 weight percent 
glutaraldehyde in neutral phosphate buffered isotonic 
35 saline for 12 hours with constant agitation at 4 degrees 



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° centigrade. At the end of the incubation period, the 

particles are filtered from the crosslinking solution and 
washed once with phosphate-buffered isotonic saline. The 
DBM particles prepared are dried under sterile conditions 
and then sterilized by an appropriate method, such as 
5 ethylene oxide, gamma, radiation, or electron beam 
sterilization. 

These activated particles may be placed directly 
in an osseous defect or alternatively, complexed with an 
organic biopolymer as described in later Examples. 
10 EXAMPLE TWO 

The demineralized bone matrix particles are 
extracted with a chaotropic agent to remove all bioactive 
or immunologic elements. Allogenic or heterogenic 
particles treated in this fashion make excellent delivery 
15 particles for the complexation of drugs, peptides, or 

proteins. After swelling in acid or alkaline solutions 
the extracted demineralized bone particles are immersed in 
the agent to be bound and released from the particle. The 
particle is then dried and crossl inked in a controlled 
20 fashion as described in Example One. The specific 

illustration below describes the use of this method. 

Ten grams of demineralized bone matrix 
particles, with a particle size of from 75 to 400 microns 
(preferably from 150 to 400 microns) , are immersed in 
25 guanidinium hydrochloride buffered with 50 millimolar 

phosphate buffer, pH 7.4, The particles are maintained in 
this extraction medium at 4 degrees centigrade for 10 to 
15 hours with gentle agitation. Optionally, protease 
inhibitors such as 0.5 -millimolar phenylmethyl-sulf onyl 
30 fluoride, 0.1 molar 6-aminohexanoic acid, are added to the 
extraction medium. 

At the end of the extraction period, the 
extracted demineralized bone matrix particles are removed 
from the extraction solution by vacuum filtration or 
35 centrifugation at 800 to 1000 rpm. The extracted 



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10 



demineralized bone matrix particles (EDBMP) are washed 10 
to 20 times with neutral sterile phosphate buffered 
saline. The particles are then dialyzed against several 
changes of neutral phosphate buffered saline to remove any 
remaining amounts of the chaotropic agent. 

A suitable bioactive peptide or protein may be 
absorbed onto EDMB particles. In this Example 
thyrocalcitonin is used in this fashion. A one gram 
fraction of the EDBM particles are immersed in a 100 ppm 
solution of thyrocalcitonin in sterile normal saline. The 
particles are maintained in this solution for 24 to 72 
hours with periodic gentle agitation. 

The complex EDBM- thyrocalcitonin particles are 
separated by vacuum filtration and rinsed once to remove 
any excess peptide. The EDMB- thyrocalcitonin particles 
15 are immersed in a low concentration glutaraldehyde 

crosslinking solution as described in Example One. The 
particles are dried and sterilized as described in that 
example. When tested in- vitro and in-vivo, particles 
showed a time dependent release of the peptide. 
20 Other peptides and proteins, such as Bone 

Morphogenetic Protein, Insulin- like growth factor. 
Epidermal Growth Factor, Nerve Growth Factor, Human Growth 
Hormone, Bovine Growth Hormone, or Porcine Growth Hormone, 
are several examples of peptides or proteins that can be 
25 carried by the EDBM matrix particles. Conventional drugs, 
such as tetracycline or other antibiotics, may also be 
delivered via this system. 

F.X&MPLE THREE 

Protein-based microcapsules can be fabricated 
30 and then partially crosslinked under controlled conditions 
so that they become reactive and bind to an organic 
biopolymer matrix under controlled conditions. As an 
illustration, a gelatin -protein microcapsule is fabricated 
and partially crosslinked to surface activate the 
35 microcapsule. 



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0 Two and one-half grams of U.S. P. gelatin and 25 

milligrams of Bone Morphogenetic Protein (purified as 
described by Urist in the above) are mixed in 8 
milliliters of sterile distilled water at 60 degrees 
centigrade. Following solubilization of the gelatin and 

5 complexation with Bone Morphogenetic protein (BMP) , 2 

milliliters of 1 millimolar phosphate buffer, pH 7.4 is 
added to the gelatin-BMP solution with constant stirring. 
This solution is maintained at 55 to 60 degrees 
centigrade. In a separate container, one hundred 

10 milliliters of an oil phase is prepared by combining 20 
milliliters of petroleum either with 80 milliliters of 
mineral oil. This solution is heated to 55 to 60 degrees 
centigrade. 

The gelatin- BMP solution is added to the oil 

15 phase with rapid stirring over a 15 second period leading 
to the formation of gelatin-BMP microspheres. Upon 
chilling to 2 to 4 degrees centigrade, the gelatin-BMP 
spheres jelled into beads. The oil phase of the solution 
is removed by vacuum filtration. The beads were washed 

20 with petroleum ether and diethyl ether. 

The microspheres so obtained are then 
crosslinked as described in Example One. In this Example, 
the microspheres are crosslinked in .03 weight percent 
glutaraldehyde in neutral phosphate buffered isotonic 

25 saline. The microspheres are filtered by vacuum 

filtration and rinsed once with neutral sterile isotonic 
saline. The spheres are dehydrated and stored dry. 
Alternatively, the spheres may be complexed with an 
organic biopolymer matrix to form a stress -bearing 

30 bioprosthesis . 

ffiY&MPLE FOUR 
Ten grams of milled bone powder (not 
demineralized) , which has been defatted and extracted with 
an organic solvent, such as diethyl ether, is immersed in 
35 a solution of tetracycline HC1 at a concentration of from 



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° 5 micrograms per milliliter to 50 milligrams per 

milliliter. Alternatively, the milled bone powder or 
particles is first partially demineralized in a .05 to 0.3 
molar solution of HC1 at 4 degrees centigrade for from 30 
minutes to 5 hours. These partially demineralized bone 
5 particles are then contacted in a solution of tetracycline 
HC1 as specified above. 

The particles are immersed in a 10 micrograms 
per milliliter solution of tetracycline HC1 for from 1 to 
24 hours at 4 degrees -centigrade. At the end of the 
1° immersion period, the particles are rinsed once in neutral 
buffered isotonic saline. The particles are collected and 
dried or lyophilized. The particles in this instance are 
collected, dried under ambient conditions and lyophilized. 

As an additional procedure, the dried particles 
15 are partially crosslinked with glutaraldehyde as described 
in Example One. As will be described in Example 6, these 
tetracycline treated demineralized bone matrix particles 
are subjected to other means of chemical group activation 
such as via carbodiimide activation of surface carboxyl 
20 groups and reaction with an amine or diamine. 

p.y AMPLE FIVE 
Other protein containing particles are 
fabricated from pulverized reconstituted collagen 
particles. As an example, collagen- tetracycline 
25 conjugates sponges are fabricated by adding tetracycline 
HC1 to an acid solubilized reconstituted collagen 
dispersion. The final tetracycline concentration is 10 to 
50 micrograms per milliliter and the collagen 
concentration is from a .5 weight percent dispersion to a 
30 3.5 weight percent dispersion. The collagen is 

solubilized with acetate or hydrochloric acid in the acid 
range or sodium hydroxide in the alkaline range. The pH 
of the collagen dispersion is adjusted to neutrality or 
near neutrality by repeated dialysis against sterile 
distilled water or phosphate buffered saline. 



35 



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After the collagen dispersion is adjusted to 
near neutrality, the appropriate drug, peptide, or protein 
is added to the collagen dispersion and agitated to assure 
complex mixing. In this example the collagen- tetracycline 
composition is poured into a cylindrical mold and allowed 

5 to stand for 24 hours in a sterile laminar flow box to 

allow initial gellation. After gellation, the dispersion 
is placed on the minus 60 degree shelf of a lyophilizer 
and freeze-dried to form a sponge material. The sponge 
conjugate material is removed from the lyophilizer and 

10 placed in a controlled dry- heat oven at a temperature of 
from 45 to 80 degrees centigrade. The heat stability of 
the molecule conjugated to the collagen determines the 
appropriate temperature. The dried sponge is removed and 
milled to a powder in an A-20 mill. The collagen- 

15 tetracycline particles produced are then surface activated 
and partially cross linked. 

EXAMPLE SIX 

The binding and covalent attachment of protein- 
based particles protein microcapsules, demineralized bone 

20 matrix particles, or protein conjugated inorganic 

particles, are enhanced by increasing the number of 
surface binding sites. This increase in binding sites 
accomplished by the following procedure. 

Ten grams of demineralized bone matrix particles 

25 are obtained with a particle size of from 50 to 400 

microns. The particles are immersed in a water soluble 
carbodiimide, l-ethyl-3- (3-dimethylaminopropyl) 
carbodiimide is varied between 0.005 molar to about 0.1 
molar preferably about 0.05 molar to about 0.1 molar 

30 preferably about 0.05 molar in a isotonic salt solution. 

The pH of the carbodiimide solution was maintained between 
about 4.7 and about 5.2 by the addition of HC1. Ethanol 
and other organic compounds, such as mannitol are added 
from time to time to alter the dielectric constant of the 

35 crosslinking solution. Alternatively, the ionic strength 



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° is increased by the addition of NaCl from about .1 molar 
to 1.0 molar. Similar modification is undertaken from 
time to time with the glutaraldehyde crosslinking 
procedures . 

The reaction with the carbodiimide proceeds from 
5 about 20 minutes up to 12 hours or more. In this 

particular example, the reaction time is 2 hours and the 
reaction is carried out at four °C, the surface activated 
demineralized bone particles are then contacted with an 
amine or diamine. Materials with amine functional groups 
10 include amino acids, poly amino acids, globular proteins 
such as albumin and gelatin, fibrillar proteins such as 
collagen and elastin. Alternatively, in this instance a 
diamine, namely hexanediamine, is used to react with the 
carbodiimide activated particles. The hexanediamine 
15 permits the increase of free available amine binding sites 
for activation by glutaraldehyde. The hexanediamine 
solution contains from .01 weight percent to about 2.0 
weight percent diamine. The optimal diamine concentration 
is approximately .1 to .5 weight percent in a neutral 
20 buffered saline solution at pH 7.4. The contact time is 
from 2 to 10 hours with the usual time being four hours. 

The particles are removed from the diamine 
solution by filtration and are rinsed several times with 
neutral buffered saline to remove excess diamine. The 
25 demineralized bone particles are added to a crosslinking 
solution of glutaraldehyde with an aldehyde concentration 
of from .001 weight percent to .25 weight percent. The 
method used is identical to Example One and the 
concentration of glutaraldehyde is .05 weight percent. 
30 The partial cross -linking occurs at 4° C in a neutral 
buffered isotonic saline solution. The crosslinking 
solution time is 8 to 12 hours. The particles filtered 
from the solution and are washed once with buffered 
neutral isotonic saline. The particles are dried and at 
35 this point can be used for binding in an organic 



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biopolymer matrix to produce a stress-bearing bone graft, 
as described herein. Alternatively, the particles are 
lyophilized and sterilized by either ethylene oxide, 
liquid sterilizing solution, gamma radiation, or. electron 
beam sterilization. 
5 EXAMPLE SEVEN 

An aqueous collagen dispersion is made from a 
high purity, medical grade, sterile powdered collagen. 
The constituted collagen dispersion is made at 2.5 weight 
percent collagen by solubilizing the collagen powder in a 
10 .01 N acetic acid buffer. The collagen powder is added, 
from time to time in concentrations ranging from 0.5 
weight percent to 2.5 weight percent. Other organic 
acids, such as lactic acid or inorganic acids, such as 
hydrochloric acid, are also used from time to time to 
15 facilitate the swelling of the collagen matrix. 

The acid dispersion of the collagen is mixed 
with moderate agitation and stored overnight to permit 
thorough swelling of the collagen gel. The collagen 
dispersion is vigorously agitated and sheared in a Waring 
20 Blender under medium to high speed using 3 to 5 

intermittent, 30 second mixing periods. The collagen 
dispersion is then poured into an appropriately sized 
centrifuge tubes and centrifuged at 800 rpm to remove 
entrained air within the collagen dispersion. The 
25 dispersion is then dialyzed against a solution of sterile 
distilled water. The collagen dispersion is repeatedly 
dialyzed against fresh exchanges of sterile distilled 
water until the pH of the collagen dispersion is in the 
range of pH 5.3 to 7.0. On occasion to obtain a 
30 dispersion with a pH of from 6.8 to 7.6 in an efficient 
manner, the collagen dispersion is dialyzed against a 
buffer solution such as neutral phosphate buffer. The 
dialyzed collagen dispersion is collected and placed in a 
container at 4 degrees centigrade. The dispersion serves 
35 as a matrix material. 



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° Two types of demineralized bone matrix particles 

are utilized in this procedure. The first type are normal 
demineralized bone particles without surface activation 
with glutaraldehyde. The second type are particles of 
demineralized bone matrix identical to the first group 

5 except they are activated by partial crosslinking in 
glutaraldehyde as described in Example One.. These two 
systems are describes as follows: 

(1) Demineralized bone particles at 85 weight 
percent are dispersed in the aqueous collagen matrix; 

10 placed in a cylindrical mold and cast by forced air 
dehydration at ambient conditions. The conjugate 
cylinders are retained for physical testing. 

(2) Demineralized bone particles, identical to 
about (1) are activated in glutaraldehyde as described in 

15 Example One. These particles are then dispersed at 85 
weight percent in the aqueous collagen matrix. The 
conjugate is placed in a cylindrical mold and cast by 
forced air dehydration at ambient conditions. The 
conjugate cylinders are retained for physical testing. 

20 To better understand the action of 

glutaraldehyde in these matrix particle conjugates, three 
other methods of addition of 0.5 weight percent 
glutaraldehyde are also employed. These are 

(3) Demineralized bone particles at 85 weight 
25 percent are dispersed in the collagen matrix. Neutral 

buffered glutaraldehyde is added to the aqueous dispersion 
so that the final concentration is 0.5 weight percent. 
The conjugate is placed in a cylindrical mold and cast by 
forced air dehydration at ambient conditions. The 
30 conjugate cylinders are retained for physical testing. 

(4) Neutral buffered glutaraldehyde is added to 
the collagen dispersion prior to the addition of 
demineralized bone matrix particles (unactivated) . The 
glutaraldehyde is added so that its concentration with 

35 respect to the total weight of the conjugate would be 0.5 



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10 



15 



20 



25 



30 



weight percent. The demineralized bone matrix particles 
are then added with mixing at a weight ratio of 85 weight 
percent. The conjugate is placed in a cylindrical mold 
and cast by forced air dehydration at ambient conditions. 
The conjugate cylinders are retained for physical testing. 

(5) Conjugate cylinders are fabricated as 
described for System (1) above, but are then immersed in a 
neutral buffered solution of 0.5 weight percent 
glutaraldehyde at 4 degrees centigrade for 72 hours. The 
cylinders are removed and washed repeatedly in neutral 
phosphate-buffered isotonic saline. The cylinders are 
replaced in their original molds and dried by forced air 
dehydration under ambient conditions. The conjugate 
cylinders are retained for physical testing. 

The following table displays the results 
obtained with the physical testing of the different 
systems. The cylinders are tested for diametrial tensile 
strength in an Instron Tester at constant loads 5 or 20 
kilograms, depending on the strength of the material. The 
dimensions of the cylinders are measured prior to testing 
and all cylinders are tested on their sides as is usual 
for the diametrial internal cohesive strength of a 
material . 

SYSTEM 



Force 

Applied 

Strain 
Profile 



I 

5Kg 



Sponge - 
like 



1 
20Kg 



3 
5Kg 



Resistant Sponge - 
to load like 
with yield 
point 



1 

5Kg 



Sponge - 
like 



5 
5Kg 



Sponge - 
like 



<2.5 psi 90 psi 



<2.5 psi <2.5 psi <2.5 psi 



35 



Diametrial 
Tensile 
Strength 

ff 0te ; collagen-demineralized bone particle 

compositions at or above 90 weight percent bone 



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particles to collagen fail to aggregate into a 
cohesive mass and spontaneous disintegrate under 
any degree of force. 

EXAMPLE EIGHT 

The nature of the matrix biopolymer also has a 
5 definite effect on the internal cohesive strength of the 
material and its ultimate strength properties. The 
procedure below illustrates the fabrication of a collagen- 
based material which is adhesive to itself or other bone 
compositions, is hemostatic, and is osteogenic. 
10 Ten (10) grams of sterile collagen powder 

(Collastat) is mixed in 100 milliliters of .IN HC1 with 
stirring-bar agitation. After 15 minutes of agitation, 
collagen dispersion is diluted from 10 weight percent to 5 
weight percent by a two- fold dilution with sterile 
15 distilled water. This results in a final acid 

concentration of .05 N HC1 and a final pH of 4.1 to 4.3. 

Four point three (4.3) grams of milled 
demineralized bone powder (particle size 125 microns or 
less; MW 0.250 sieve) are added to the collagen mixture. 
20 After thorough stirring the 5 percent dispersion is mixed 
in a Waring Blender for 5 to 10, 20 second agitations to 
increase the dispersion viscosity. The thickened solution 
is poured into centrifuge tubes and spun in a table- top 
centrifuge at 400-600 rpm for 5 minutes to remove air and 
25 concentrate the collagen. 

Excess fluid supernatant is removed by pipetting 
and the collagen conjugate fraction is collected into a 
single volume (approximately 170 milliliters) . This 
collagen-demineralized bone dispersion is stored at 4 
30 degrees centigrade for at least one hour to check for 

consistency and the presence of phase separation. The pH 
of the mixture is 4.50 to 4.57. 

The collagen mixture is transferred to dialysis 
tubing (Spectrapor. 12,000 to 14,000 molecular weight 
35 cut-off) and dialyzed overnight against sodium phosphate 



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buffer .02 molar pH 7.4. The collagen-DBP dispersion is 
removed from the dialysis tubing using aseptic technique. 
The dispersion is homogeneous and shows no evidence of 
separation. The pH of the dialyzing solution is 6. 5. 
The pH of the collagen dispersion is 5.00 to 5.12. 

5 The dialyzed collagen-DBP dispersion is 

collected, placed in a 250 milliliter centrifuge bottle, 
then spun at 800 rpm for 10 minutes. The clear 
supernatant is collected and checked for pH which is 5.10. 

The collagen-DBP dispersion is placed in sterile 

10 petri dishes and frozen, under aseptic conditions, at 

minus 4°C under vacuum, the vacuum is maintained for 18 to 
24 hours to assure complete dehydration. The resultant 
foam- like sponge material is placed in an A- 10 mill and 
milled into a powder. The powder is divided into equal 

15 aliquots and bottled. The bottles of collagen-DBP powder 
are sterilized under ethylene oxide for 2 and 1/2 hours. 
The bottles are aerated under vacuum for at least 24 hours 
and then sealed under vacuum. 

The resultant material is hemostatic in that it 

20 promotes the clotting of blood. 

EXAMPLE NINE 

The collagen-demineralized bone particle powder, 
as described in Example Eight is reconstituted in a 5 mM 
solution of sodium phosphate buffer, pH 8.0. 

25 Approximately .2 grams of the powder is hydrated with 1 
milliliter of the buffer and mixed to assure complete 
mixing. Demineralized bone particles, average particle 
size 250 microns are activated and partially crosslinked 
as described in Example One. A weight of .10 grams of 

30 these particles are added to the buffer- collagen conjugate 
dispersion with gentle mixing. The mixture is placed in a 
cylindrical mold and dehydrated by forced air under 
ambient conditions. The resultant disc dried very 
rapidly, i.e., within 4 to 10 hours. If the mass is 

35 lyophilized, a more porous structure results. The detail 



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° of the mold is well reproduced on the cylinder. Cylinders 
demonstrate a smooth surface appearance and have 
sufficient integrity to be milled or ground to precise 
shapes with surgical burs or grinding wheels in low or 
intermediate speed handpieces. The cylinders so produced 

5 are tested for diametrial tensile strength at 20 kilogram 
constant load. The results are as follows: 

gYSTEM 6 

pnmp Applied 20 kg load 

si- re in Profile Linear, elastic 

10 behavior with 

increased module 
in tension 

Diametrial Tensile 

■^rpnath (PSD 279 to 320 psi 

15 EXAMPLE TEN 

Other drugs, proteins, or peptides are added to 
the matrix phase of these compositions which contain ac- 
tivated particles. For example, a purified or recombinant 
bone morphogenetic protein, as described by Urist in U.S. 

20 Patent 4,294,753 is added to the matrix prior to the 

addition of activated particles or microcapsules. As the 
stability of the conjugate does not rely on addition of 
glutaraldehyde to the bone matrix, the chance of 
inactivating the BMP molecular is reduced. The conjugate 

25 material can be used in its aqueous form, however, in this 
instance the activated demineralized bone 
particles-collagen-BMP conjugate is dehydrated under 
ambient conditions, as described earlier. Another sample 
is dehydrated and then lyophilized at minus 40 to minus 60 

30 degrees centigrade. 

Another conjugate, made in identical fashion 
with respect to order of addition of components, consist 
of activated demineralized bone particles -collagen and 
tetracycline HCL. This conjugate is dehydrated and 

35 lyophilized. Other proteins and peptide growth factors 



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are evaluated when complexed with the matrix phase of this 
novel, cohesive compositions. 

EXAMPLE ELEVEN 

The activated and partially crosslinked protein 
particles, microcapsules or demineralized bone matrix 
5 particles whose methods of surface activation were 

described in above Examples, are added to viscous mixtures 
of blood proteins, glycoproteins, or cell component 
fractions . 

Specifically, 0.5 grams of activated 

10 demineralized bone matrix or bone matrix particles are 

removed from the container in which they are sterilized. 
In this instance, the bone is being used to fill an 
osseous defect in a laboratory animal. Five milliliters 
of the animal's blood is withdrawn by venipuncture, the 

15 blood is spun at 800 to 1000 rpm in a table-top centrifuge 
to spin- down platelets, white blood cells and red blood 
cells. The blood is drawn into a plain vial which does 
not contain any type of anticoagulant. After the cellular 
components of the blood are pelleted, the supernatant 

20 containing serum is withdrawn carefully with a pipette. 
The serum is added to the activated demineralized bone 
particles so that the particles are evenly coated. The 
ratio of activated bone particle to serum or plasma can 
vary from 20 to 95 percent by weight. The conjugate is 

25 placed into the bony defect such that it is filled 

completely. The defect is gradually replaced with new bone 
over a period of 6 to 12 weeks. 

The identical procedure is undertaken with 
another research animal except this time the blood is 

30 drawn into a heparinized tube and plasma is obtained after 
centrifugation. This blood plasma is combined with the 
activated blood particles in a manner identical to the 
above. 

In certain instances, such as large osseous 
35 defects or non-unions, it is beneficial to add bioactive 



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° molecules or antibiotics to the serum or plasma fraction. 
Rabbit bone morphogenetic protein is purified from rabbit 
demineralized bone matrix, using a method described by 
Urist in U.S. Patent 4,294,753. The purified BMP is added 
to the plasma so as to constitute about .5 to 3 percent by 
5 weight. After mixing the lyophilized protein into the 
plasma and dispersing it thoroughly, the activated 
demineralized bone particles are mixed into the BMP-plasma 
at a weight ratio of 80 to 90 parts of particles to 10 to 
20 parts of plasma. 
10 Another laboratory animal is presented with a 

bone injury with possible bacterial contamination. Blood 
is drawn and plasma obtained as previously mentioned. To 
the plasma is added a powder tetracycline hydrochloride 
salt at a concentration of 5 to 25 micrograms per 
15 milliliter. The antibiotic is mixed thoroughly in the 

plasma and the plasma mixed with activated demineralized 
bone particles at a weight ratio of 80 to 90 parts 
particles to 10 to 20 parts plasma -tetracycline. 

EXAMPLE TWELVE 

20 The proteins which constitute the matrix can be 

further modified by the addition of phospholipids. In 
particular, reconstituted collagen and acidic 
phospholipids demonstrate together an enhanced uptake of 
calcium as compared to collagen matrixes without 

25 conjugated acidic phospholipids. 

A 2.5 weight percent collagen dispersion at a pH 
of 5.0 to 5.5 was used for the addition of an acidic 
phospholipid, L-alpha-phosphatidic acid, dipalmitoyl, is 
added to the above reconstituted collagen dispersion at 

30 from .01 milligrams per milliliter collagen to 10 
milligrams per milliliter collagen. The conjugate 
dispersion is dehydrated at ambient temperatures and 
lyophilized. Alternatively, activated protein particles, 
microcapsules, or demineralized bone matrix particles are 

35 



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added to the conjugate aqueous dispersion as described 
within this disclosure. 

EXAMPLE THIRTEEN 
A reconstituted collagen matrix can be further 
modified by the addition of an alkaline source of calcium 
5 ions. For example a reconstituted collagen dispersion 
with a collagen composition of 0.5 to 2.5 percent by 
weight and a pH of 5.0 to 5.5 is dialyzed against a 
saturated solution of calcium hydroxide in sterile 
distilled water. When the pH of the collagen dispersion 
1° reaches 10 to 10.5 the collagen dispersion is removed from 
the alkaline solution, placed in an appropriate sized mold 
and lyophilized to form a sponge. Another aliquot of the 
collagen- calcium hydroxide is combined with activated 
demineralized bone particles and mixed to thoroughly 
15 disperse the particles in the alkaline matrix. The 
conjugate is dehydrated and lyophilized to form a 
stress -bearing sponge material. 

These collagen- calcium hydroxide conjugates 
demonstrate rapid release of the calcium and hydroxide 
20 ions and load only sufficient amounts of hydroxide ions to 
slightly adjust the pH. 

EXAMPLE FOURTEEN 
A calcium hydroxide (CaOH)/ collagen- gelatin 
microbead is fabricated using the following method. A 

25 reconstituted collagen dispersion at neutral or acidic pH 
is made as described in prior Examples. Powdered calcium 
hydroxide is slowly added to the dispersion until a pH 
such that a collagen to gelatin conversion was evident. 
The pH necessary to effect this conversion is 

30 approximately 11.0 or above. The visual effect at this 
conversion was quite noticeable, as the collagen 
dispersion loses all its translucency and becomes opaque 
and chalky. 

The colloidal dispersion can be formed into 
35 microbeads by immersion in an oil phase, as described in 



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Example Three. Nevertheless , in this example, the 
collagen- CaOH gelatin dispersion may be dried by 
lyophilization at minus 40 minus 60 degrees centigrade. 
Dehydration at ambient temperatures also yields a solid 
mass . 

5 This mass is milled and pulverized is into fine 

particles. The particles are partially cross-linked in a 
.05 weight percent glutaraldehyde solution at a pH of 7.8. 
After rinsing once, the activated collagen/gelatin- CaOH 
particles are added to an alkaline collagen dispersion 
10 containing calcium hydroxide. This mixture may be 
lyophilized or dehydrated. However, activated 
demineralized bone particles may be added in a weight 
percent range of from 10 to 85 weight percent. 

EXAMPLE FIFTEEN 
15 A collagen- calcium phosphate conjugate is 

derived as described by Cruz in U.S. Patent 3,767,437. A 
reconstituted collagen dispersion at a pH of 3.5 to 4.5 in 
sodium acetate is dialyzed first against 3 to 7 changes of 
deionized water and then dialyzed against a saturated 
20 solution of calcium hydroxide for 2 to 5 changes. The 

collagen-CaOH solution is then dialyzed against a solution 
of phosphoric acid adjusted to pH 3 .0 to 4.0. The 
dialysis for 2 to 6 changes resulted in a Collagen- Calcium 
Phosphate conjugate. The dispersion is lyophilized or 
25 dehydrated under an ambient conditions . The resultant 
mass is pulverized under moderate force. The resultant 
particles are sieved to a uniform particle size of 50 to 
1000 millimicrons. The particles are dried and placed in 
a .08 glutaraldehyde solution also contains 8 mM calcium 
30 phosphate buffer. The particles are filtered and rinsed 
once with sterile distilled water. 

The partially crosslinked, activated particles 
are added to a reconstituted collagen dispersion with 
moderated mixing and agitation. The dispersion can be 
35 left in a viscous gel -state, lyophilized, or dehydrated at 



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ambient conditions. The resultant dried mass has a 
diametrical tensile strength greater than one hundred PSI. 

EXAMPLES SIXTEEN - 
Collagen- calcium phosphate particles, prepared 
and activated as described in Example Fifteen, are added 

5 to a composition derived as described in Example Seven, 
System No. 2. Inorganic particles are added to collagen 
matrix phase, so that no more than 20 weight percent of 
the entire conjugate is composed of the protein/ inorganic 
particles. The entire mass is cast and dehydrated as 

10 described in the earlier Examples. 

EXAMPLE SEVENTEEN 

Collagen- calcium phosphate particles, prepared 
and activated as described in Example Fifteen are added to 
a composition derived as described in Example Nine. The 

15 inorganic particles are added so that no more than 20 

weight percent of the entire conjugate is composed of the 
protein/ inorganic particles. The entire mass is cast and 
dehydrated as described in the above Examples. 

KX AMPLE EIGHTEEN 

20 collagen- calcium phosphate particle conjugate 

derived from either hydroxyapatite or tricalcium phosphate 
particles even when crosslinking agents such as 
glutaraldehyde in low concentrations are added to the 
collagen matrix, demonstrate very low tensile strengths 

25 i.e., on the order of 30 psi or less. A method is 
described in this example to provide 

collagen- hydroxyapatite or collagen- tricalcium phosphate 
conjugates with enhanced strength and reduced plucking of 
the inorganic particles from the matrix. 

30 An acid dispersion of reconstituted collagen is 

made in the acid pH range using 0.05 acetic acid as de- 
scribed earlier. The collagen dispersion is made at .75 
weight percent collagen sheared in a Waring Blender and 
dialyzed against sterile isotonic saline until the pH of 

35 the dispersion reaches a range of 4.0 to 5.5. Tricalcium 



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10 



phosphate particles medical grade and sterile with a 
particle size of 50 to 150 millimicrons are added to the 
dispersion with moderate mixing. The dispersion is 
degassed under vacuum with moderate agitation. The 
dispersion is placed in a dialysis tube and dialyzed 
against .01 molar phosphate buffer at pH 8.0. The 
dialysis tube is periodically removed aseptically and 
inverted several times to prevent separation of the 
mineral phase. After 24 to 48 hours of dialysis the 
dispersion is removed from the dialysis tubing , poured 
into a stainless steel mold and lyophilized at between 

minus 40 and minus 60°C. 

At the conclusion of lyophilization the sponge 
like mass is cut into about .5 cm square cubes and milled 
carefully at low settings in an A- 10 mill so as to provide 

15 a group of collagen -mineral particles on order of about 
250 to 550 microns. The particles are activated in a 
manner consistent with one of the embodiments of the 
invention. Specifically, in this example, the conjugate 
particles are immersed in a neutral buffered isotonic 

20 solution of bout 0.08 weight percent glutaraldehyde . The 
concentration of the glutaraldehyde was varied from .001 
to .25 weight percent glutaraldehyde. The conjugate 
particles are activated for about 8 to 12 hours at 4 
degree centigrade. The particles are removed by vacuum 

25 filtration and washed once in neutral buffered isotonic 
saline. 

The activated protein- coated mineral particles 
are added to a reconstituted collagen dispersion of one to 
2.5 percent by weight collagen, with a pH of from 3.5 to 
5.0. The activated particles are added to the dispersion 
in a weight range of from 25 to 85 percent by weight. The 
preferred range is from 40 to 75 percent by weight. The 
activated protein-mineral particle/reconstituted collagen 
conjugate is poured into a stainless steel mold and dehy- 
drated at ambient temperatures with forced recirculated 



30 



35 



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° air. The conjugate, once dehydrated may be lyophilized at 
minus 40 to minus 60 °C. 

Another conjugate of this type is cast except 
that prior to dehydration, a bioactive protein, peptide, 
or drug is added to the matrix, as has been described in 

^ earlier Examples. 

EXAMPLE NINETEEN 
While a stable coating of reconstituted collagen 
can be formed in a continuous adherent layer on the . 
surface of an inorganic particle a preferred method is to 
1° form multiple chelation links between the calcium* rich 
surface and the protein-based surface layer. 

Particles of a calcium phosphate ceramic 
material, namely tricalcium phosphate particles with a 
size of about 100 millimicrons are immersed in a 10 ppm 
15 solution of L-y-carboxyglutamic acid. The particles are 
incubated in this solution for 24 to 48 hours 4°C. The 
particles are removed from the solution dried under 
ambient conditions and immersed in about a 0.5 to 1 weight 
percent collagen dispersion containing about 10 to 50 ppm 
20 of L-y-carboxyglutamic acid. The particles are agitated 
gently in this dispersion filtered from the dispersion 
then placed in a .15 molar NaCl solution containing .05 
molar sodium phosphate buffer adjusted to pH 7.4 with 
dibasic and tribasic sodium phosphate. After 15 minutes 
25 to one hour in this solution. The collagen coated 

particle is partially crosslinked in a .075 weight percent 
solution of glutaraldehyde for 8 to 10 hours. 

The particles are removed from the 
glutaraldehyde solution by filtration then rinsed once in 
30 sterile saline solution. Once activated some of these 
particles are used directly is osseous defects. 
Alternatively, some of the activated particles are mixed 
into a l weight percent dispersion of the reconstituted 
collagen. The particles are mixed and agitated to assure 
35 a uniform dispersion. The gel so obtained is used in 



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certain osseous defects. Alternatively, the collagen- 
particle dispersion is lyophilized or dehydrated under 
forced air under ambient conditions. The resultant 
material is sterilized with ethylene oxide, gamma 
radiation, and/or by immersion in a .2 percent buffered 
^ glutaraldehyde solution. 

EXAMPLE TWENTY 
In place of the L-y-carboxyglutamic acid 
disclosed in Example Nineteen, the sodium salt of poly-L- 
glutamic acid or the random copolymer of L-glutamic acid, 
10 which contains at least one lysine in its repeating 
structure, may be used to coat the calcium phosphate 
particle prior to complexation with reconstituted 
collagen. In this procedure, the particles are mixed and 
agitated within the polyamino acid solution, then under 
15 ambient conditions the particles are dehydrated or 

alternatively, lyophilized. The coated particles are 
mixed in a reconstituted collagen dispersion and again 
dried to provide a uniform coating. The coated particles 
so produced are partially crosslinked in .05 weight 
20 percent neutral buffered glutaraldehyde for about 10 to 12 
hours at 4°C. The particles are vacuum filtered from the 
activating solution and dried. The particles are then 
used as described within the embodiments of the invention. 
Alternatively, the polyamino acid coated particles once 
25 dried may be added to a reconstituted collagen dispersion 
which contains about .05 to .1 weight percent 
glutaraldehyde. The entire conjugate may be dehydrated or 
lyophilized, then milled to a powder if further 
complexation is intended. 

30 EXAMPLE TWENTY -ONE 

System No. 2 of Example Seven described the 
fabrication of a reconstituted collagen/ activated 
demineralized bone matrix conjugate with improved internal 
cohesive strength. The weight percentage of activated 
35 particles is demonstrated to be useful in the range of 5 



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to 85 weight percent of the conjugate. Nonactivated 
particles can be added to matrix in weight percent ranging 
from 0 to 95 percent of the total conjugate weight. If 
the non-activated or activated particles are inert, 
inorganic particles, specifically, tricalcium phosphate 
5 hydroxyapatite, their weight percent does not exceed 20 
weight of the total conjugate mass. 

EXAMPLE TWENTY -TWO 

Example Nine described a cohesive stress -bearing 
conjugate which is composed of an adhesive 
10 collagen- demineralized powder which is hydrated and 

admixed with an additional 20 weight percent of activated 
demineralized bone particles. This composition is 
comprised of 30 weight percent original unactivated 
particles plus twenty weight percent activated 
15 demineralized bone particles (average particle size 150 

microns) . The percentage of activated demineralized bone 
particles is from time to time, increased up to 50 weight 
percent of the total mass. Other conjugates are admixed 
to contain up to 20 weight percent (with respect to the 
20 total conjugate mass) of activated or non-activated inert 
inorganic particles consisting of particles of tricalcium 
phosphate or hydroxyapatite with a particle size range of 
20 to 750 millimicrons, with the preferred range being 20 
to 150 millimicrons the total weight percent of particles 
25 of any type greater than 85 percent of the total mass. 

EXAMPLE TWENTY -THREE 
The matrix component of the above examples may 
contain from a non- fibrillar collagen group, such as gela- 
tin. Sufficient gelatin with a Bloom strength of at least 
30 200 is added to the reconstituted collagen so that no more 
then 10 weight percent of matrix consists of gelatin. 

EXAMPLE TWENTY FOUR 

Polyamino acid microcapsules may be used to form 
protein-based, partially crosslinked particles as 
35 described in Example Three. The same procedure is 



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followed except that a viscous solution of poly- 1- lysine 
is used instead of gelatin. The other exception to the 
procedure is that the poly-L- lysine is used instead of 
gelatin. The other exception to the procedure is that the 
poly-L- lysine is warmed only to 37 to 43 degrees 
5 centigrade. 

EXAMPLE! TWENTY -FIVE 

Other types of inorganic particles can be 
activated and reacted with collagen, gelatin, polyamino 
acid or polyalkenoic acids to form rigid, stress -bearing 
10 implants and cements. Aluminosilicate glasses, which 

contain varying amounts of calcium fluoride, are used for 
stress -bearing cements and implantable bone replacement 

structures. 

These hard- setting cements formed from the 
15 reaction of powders and liquids. Specifically, milled 
aluminosilicate glass, designated G-309 or G-385 are 
provided. The reactant liquid consists of from 35 to 55 
percent polyacrylic acid, molecular weight from 15,000 to 
60,000 and from 2 to 35 weight percent reconstituted 
20 collagen and the balance distilled, deionized water. 

The powder and liquid are mixed at a powder to 
liquid ratio of from 1.4 to 3 grams per milliliter liquid. 
The working time for the cement is about 1 minute 45 
seconds to 2 minutes 45 seconds and the final set from 5 
25 minutes 30 seconds to 6 minutes 45 seconds.. 

EXAMPLE TWENTY SIX 

The reconstituted collagen-glass ionomer cements 
are varied by the addition of from .01 to 3 percent 
glutaraldehyde into the liquid component as described in 
30 Example Twenty-Six. The inclusion of glutaraldehyde 

shortens the working/setting time and produces a stronger 
cement as determined by physical testing. 

EXAMPLE TWENTY SEVEN 

The liquid component as described in Examples 
35 Twenty- Five and Twenty Six can be further -modified by the 



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addition or substitution of polyamino acids for the 
polyalkenoic acids in the liquid component. For the 
entire polyacid component of the liquid may be replaced 
with poly- L- glutamic acid. Alternatively, from 5 to 45 
weight percent of the liquid component may consist of a 
5 polyamino acid, namely, poly -L- glutamic acid, 

poly-L-asparatic acid, poly- L- lysine, homopolymers or. 
random co- polymers of these or any polyamino acid may be 
added to the liquid component combinations of these 
polyamino acids polymers vary the setting time and the 
10 ultimate physical strength of the cement or implant* 

EXAMPLE TWENTY EIGHT 
Bone Morphogenetic Protein and/or bone proteins 
extracted from demineralized bone matrix may be 
incorporated into uniform unilamellar liposomes for 
15 controlled delivery to osseous defects. The procedure for 
incorporation of the bioactive proteins onto and into the 
membrane bilayer is described below. 

A phospholipid, 
l-palmitoyl-2-oleoyl-phosphatodylchlorine, is dispersed in 
20 an aqueous (sterile distilled water) phase by sonication 
and then mixed with lyophilized BMP such that the protein 
to lipid mass ratio to produce unilamellar BMP liposomes 
of optimal size (high encapsulation efficiency) is in the 
range of 1:2 to 1:3 with the optimal ratio being 1:2.5. 
25 The resultant mixture is dried under nitrogen in 

a rotating flask. The dried sample is then rehydrated in 
aqueous medium under nitrogen with gentle rotation of the 
flask. The resulting unilamellar liposomes where 
separated from the free morphogenetic protein by 
30 chromatography through a B-4 or G200 Sephadex column. 

The BMP- liposomes are stored at 4°C or 
alternatively, lyophilized. Prior to implantation 
reconstituted collagen sponges allogenic bone autogenous 
bone grafts or demineralized bone matrix can be soaked in 
35 the liposome preparation to stimulate osteogenesis. 



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° Alternatively, the BMP- liposome can be mixed with an 
aqueous collagen dispersion for direct placement or 
injection to the wound site, or added to the matrix phase 
described in embodiments of this invention. 

EXAMPLE TWENTY - NINE 

5 Bone morphogenetic protein and/ or extracted bone 

proteins can be entrapped in the patient's own red blood 
cells by resealing the cell ghosts in the presence of the 
bioactive proteins. This permits a highly biocompatible 
delivery system for BMP delivery to a wound site. 

10 Fresh heparin- treated whole blood (about 50 

milliliters) is centrifuged at 1000 gs for 10 minutes. 
The plasma and buffy coat is removed and the cells are 
washed three times in cold (4 degrees centigrade) Hanks 
Basic Salt Solution (HBSS) . The packed cells are mixed 

15 rapidly with twice their volume of cold hemolysing 
solution consisting of distilled water containing 
approximately .5 milligram per milliliter BMP. After 5 
minutes equilibration in the cold, sufficient concentrated 
cold HBSS is added to restore isotonicity. This 

20 suspension is warmed to 37°C and incubated at that 
temperature for 45 minutes. The resealed cells are 
collected by centrifugation at 1000 gs for 15 minutes and 
washed three times with isotonic HBSS to remove any 

untrapped enzyme. 

25 The encapsulated BMP/RBC conjugate may be 

pelleted and the pellet placed directly into an osseous 
defect. The conjugate RBCs may be surface activated and 
partially crosslinked and incorporated into an osteogenic 
and/ or stress -bearing implant. Monoclonal antibodies, to 

30 bone tissue antigenic markers, may be attached to the 

surface of the cells so that the osteogenic proteins can 
be directed, parenteral^, to an osseous defect to promote 
heating . 



35 



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0 FY AMPLE THIRTY 

The method of Example Twenty such that a calcium 
binding protein or peptide is used to create a bond 
between the inorganic particle and the matrix. A calcium 
binding peptide of molecular weight of 5,000 to 7,000, 
5 namely, osteocalcin, which binds to hydroxyapatite may be 
used as the calcium binding interface in this method. The 
particle is immersed in a 1 to 1000 ppm solution of 
osteocalcin prior to drying to affect this bound. The 
procedure in Example Twenty is then followed. 

10 EX AMPLE TH IRTY -ONE 

The substrate or matrix for the novel bone graft 
material of this invention may be, demineralized, freeze- 
dried bone allograft or matrix <DFDBA or DFDBM) , is 
processed by procedures well known in the art. By way of 
15 example, the process may include all or some the following 
steps, as' described by Mellonig (see "Freeze-Dried Bone 
Allografts in Periodontal Reconstructive Surgery," Dental 
Clinics of North America, Vol. 35, No. 3. July 1991.): 

1. Sterile harvesting of cortical bone. This bone 
20 material is sometimes placed in an antibiotic 

solution. 

2 . The cortical bone is grossly cut to particle of 
500 microns to 5 mm. Strips, wedges, chips, or 
other shapes may also be fabricated. 

25 3> The graft material is immersed in 100% ethyl 

alcohol for 1 hour to remove fat and to 
inactivate virus. 
4. The bone is then frozen at -80 degrees 

Centigrade for 1 to 2 weeks to inhibit 

30 degradation. During this time period, test 

results from serologic tests, antibody and 
direct antigen assays, and bacterial -cultures 
are obtained and bone is retained, discarded, or 
sterilized by additional methods. 



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0 5 The bone is freeze-dried to remove more than 95% 

of its water content. 
6# The cortical bone may be ground and sieved to a 

finer particle size. By way of example, about 

250 to about 750 microns. 
5 7. The -bone graft material is again immersed in 

100% ethyl alcohol and then washed repeatedly in 

distilled water to remove all prior chemicals 

used in the processing. 
8- The bone graft material is decalcified in 0.6 N 

10 HC1 to remove virtually all of the mineral 

calcium, leaving the organize bone matrix. 

9. The bone is washed in sterile water and/or 
sodium phosphate buffer to remove residual acid. 

10. The demineralized bone matrix is refreeze dried 
15 and vacuum sealed in sterile containers. 

Those skilled in the art will realized that the 
actual sequence of procedures and steps used in this 
process may vary among different tissue banks that process 
the bone graft material. This bone graft is further 
20 processed by methods and materials described below to 
produce the enhanced, osteogenic bone graft material 
described in this disclosure. 

EXAMPLE THIRTY -TWO 

Demineralized. freeze-dried, bone matrix powder 
25 (DFBM) is obtained from a tissue bank. In this example, 
DFBM powder was obtained from Musculoskeletal Transplant 
Foundation (Homdel, N. J. ) . By way of example, the 
procedure for processing of freeze-dried, demineralized 
bone matrix may involve some or all of the following 
30 steps: antibiotic soak, grinding of the bone matrix, 

washing of the ground bone matrix in sterile water and/or 
100% ethyl alcohol, demineralization in 0.5 to 0.6 N HC1, 
a sterile water rinse to remove residual HC1 acid, 
followed by ethanol wash, and the final step of freeze 
35 drying the demineralized bone matrix powder. This 



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material obtained was provided in the form of a sterile 
powder . 

The sterile freeze -dried demineralized bone 
powder was removed from its sterile glass bottle 
container, placed in a covered sterile plastic well. A 

5 saturated solution of USP calcium hydroxide is prepared in 
sterile distilled water solution. After the insoluble 
portion of the CaOH solution has sedimented to the bottom 
of the solution container, the supernatant is removed with 
a pipette and suction and placed in a separate sterile 

!0 container. 

The saturated calcium hydroxide supernatant is 
removed with a sterile syringe containing a 27 gauge 
needle. The calcium hydroxide concentration of a 
saturated calcium hydroxide solution is, according to 

15 Lange's Handbook of Chemistry, llth edition, approximately 
0.19 parts of calcium hydroxide to 100 parts of water at 0 
degrees Centigrade, or approximately 19 mg/100 mis of 
water. One of skill in the art will understand that the 
calcium concentration of this saturated solution, however, 

20 is variable, and is dependent on the temperature. The 

calcium hydroxide solution is dispensed onto the sterile 
freeze -dried demineralized bone powder until the bone 
powder matrix is visibly saturated with the solution. The 
saturated DFBPM material is permitted to dry under ambient 

25 conditions in a sterile hood. The dried, calcium- enriched 
demineralized bone matrix is lightly re-ground into a fine 
powder and replaced in a sealed sterile glass container 
until needed for implantation. By way of example the 
weight proportions or weight ratios of added salt to bone 

30 can vary from about 0.001% to about 20% by weight. By way 
of example, the weight proportion or weight ratio ratio of 
added calcium to bone can vary from about 0.001% to about 
10% by weight. 



35 



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10 



qv&MPLE TWTBTY- THREE 

The saturated calcium hydroxide supernatant is 
diluted in a series of serial dilutions to achieve varying 
dilutions of calcium salt concentration. Two solutions, 
one representing a two-fold dilution (2 to 1 dilution) of 
the saturated calcium hydroxide supernatant, the second 
representing a four-fold dilution (4 to 1 dilution) of 
that same saturated solution, are prepared in a sterile 

water solution. 

Again using a syringe with a 27 gauge needle, 
each of the two diluted calcium hydroxide solutions are 
added, respectfully, to separate l cc portions of freeze- 
dried demineralized bone powder until the bone powder mass 
appeared fully wetted and saturated with the respective 
solutions . 

15 AMPLE TFT RTY- FOUR 

The demineralized bone matrix powder 
compositions described in Examples Thirty Two and. Thirty 
Three, together with corresponding demineralized bone 
matrix powders which are from the same lot as each 
experimental batch (thus serving as the corresponding 
control groups) , were implanted intramuscularly in the 
hind thighs of laboratory mice. Each experimental batch 
of DFDBA was paired with its corresponding control 
material in a paired grouping in each animal. After 
intramuscular implantation, the paired sites were 
radiographed at 8, 29, and 46 days to assess the presence 
and size of mineralized bony masses produced through 
osteoinduction by the intramuscular implants. The animals 
were sacrificed at the prescribed time frame and implants 
with surrounding tissues were dissected and prepared for 
histologic evaluation and analysis. 

The results of the radiographic and histologic 
analysis of these DFDBA implants are described in Table I 
below: 



20 



25 



30 



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TABLE I. FREQUENCY OF BONE INDUCTION IN INTRAMUSCULAR 
IMPLANTS 



Experiment One 



10 



15 



System Tested 



Control DFDBA 

Experimental 
DFDBA (sat.) 

Experimental 
DFDBA (1:2 dp.) 

Experimental 
DFDBA (1:4 dil.) 



8 Davs 



29 Davs 



46 Davs 



Freq. Percent Freq. 

0/28 0 % 0/28 

10/10 100 % 10/10 

0/10 0 % 0/10 

0/10 0 % 0/10 



Percent Freq. 

0 % 0/28 

100% 10/10 

0 % 1/10 

0 % 0/10 



Percent 

0 % 
100 % 

10 % 

0 % 



20 



25 



30 



35 



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A second implantation experiment was undertaken 
to assess both the reproducibility of the first 
experiment, and to determine both the frequency and size 
of bone induction and mineralization in the experimental 
and control DFDBA intramuscular implants. The results ar 
depicted in Table II: 

TABLE II. FREQUENCY AND SIZE OF BONE INDUCTION IN 
" " INTRAMUSCULAR IMPLANTS 



15 



30 



10 Ex periment Two 

System Tested 8 Days 15 Da > s 

Freq . Si7£of Cal.* FjgQx Size of CaL 
Control DFDBA 0/10 0.0 0/10 0.0 

Experimental 10/10 2.45 10/10 2.45 

DFDBA 

(sat. Calcium salt) 

* Size of calcification determined by grading scale ranging from "1" to "4", with 
grade of "4" being the largest mass. 

20 

As shown in Tables I and II, data obtained at 
all time- frames evaluated revealed that the experimental 
DFDBA complexed with the saturated calcium hydroxide 
solution demonstrated 100% induction and formation of new 
25 vital bone in all intramuscular implants. The DFDBA which 
was not treated with bone in all intramuscular implants. 
The DFDBA which was not treated with the saturated calcium 
hydroxide solution failed to produce radiographically 
detectable bone, even at 6 weeks. The DFDBA bone powder, 
which was treated with a l to 2 dilution of the saturated 
calcium hydroxide solution, produced 1 out of ten implants 
with radiographically evident bone formation at 6 weeks 
evaluation. The DFDBA treated with the 1 to 4 dilution of 
saturated calcium hydroxide did not produce any 
radiographically detectable bone at the 6 week time point. 



35 



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Nevertheless, the histologic analysis of the DFDBA matrix 
powder treated with the 1 to 2 and the 1 to 4 dilutions of 
the saturated calcium hydroxide solution did provide 
beneficial cellular responses with reduced inflammatory 
cells and early evidence of an osteogenic response, when 
5 compared with the inflammatory cellular, response seen with 
the untreated, standard demineralized bone material. 

EXAMPLE THIRTY- FIVE 

The demineralized bone matrix material may be 
rinsed with a variety of other buffers or salt solutions 
10. prior to the exposure to the free calcium salt solution. 

For example, the bone matrix may be demineralized in 0.5 N 
or 0.6 N HC1 for a sufficient time period to effect 
sufficient mineral removal to demonstrate osteogenic 
properties (as measured by a residual pH level of 1.0 or 
15 less) . After removal from the acid solution, and removal 
of residual acid by washing in sterile distilled water, 
the demineralized bone matrix may be rinsed in various 
concentrations of buffer solutions, adjusted to various 
pHs as may be desired. Use of neutral or slightly 
20 alkaline buffer systems can assist in neutralizing 
residual acid left after water rinsing. 

For example, the demineralized bone powder, 
after demineralization in 0.5 or 0.6 HC1 and sterile water 
rinsing, may then be rinsed in a phosphate buffer 
25 solution, for example disodium phosphate buffer solution, 
ranging from 0.001 M to 0.2 M (pH 7.5 to 9.0) . After 
buffer rinsing, the demineralized bone matrix is then 
saturated with a solution containing soluble calcium, such 
as a saturated solution of calcium hydroxide, and then 
30 permitted to dry under ambient conditions or by 
lyophilization. 

pviMPT.K THIRTY- SIX 

The calcium source may be delivered to the 
surface of the demineralized bone matrix by a means other 
than aqueous or water solution. For example, the soluble 



35 



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calcium source may be applied in a water-soluble or water- 
insoluble film former. Alternatively, the DFDBA treated 
with the soluble calcium salt may be further combined with 
a water-soluble or water insoluble filming forming agent. 
For example, the soluble calcium enhanced 
5 demineralized bone matrix may be complex with a aqueous 
collagen dispersion of gelatin solution and, optionally, 
further lyophilized or dehydrated into a sponge of 
membrane configuration. Alternatively, the soluble 
calcium solution may be added to collagen dispersion or 
10 gelatin solution, after which untreated demineralized bone 
matrix powder then added to the calcium/ collagen or 
calcium/gelatin dispersions or solutions, and the entire 
conjugate dehydrated or lyophilized into a solid form for 
implantation. 

15 EXAMPLE TP TRTY- SEVEN 

The calcium salt saturated bone mass, processed 
as described in these examples, can be lyophilized rather 
than allowed to dry under ambient conditions. 

E y&MPT.E TH TRTY - EIGHT 

20 The demineralized bone matrix starting mat«rial 

can be processed by alternative methods which extract the 
bone matrix further to remove additional potential 
antigens. First, the bone graft is placed in a 1:1 
chloroform-methanol solution for 4 hours at 25 degrees C. 

25 A solution of 100% ethyl alcohol may be substituted for 
the chloroform-methanol solution. The bone is then 
immersed in a. 0.1 M Phosphate buffer solution, pH 7.4, 
containing 10 rriM/L. iodoacetic acid and 10 mM/L sodium 
azide for 72 hours at 37 degrees C. After rinsing in 

30 sterile distilled water, the bone graft is placed in 0.6 N 
Hydrochloric acid for 24 hours at 2 degrees C to 
facilitate demineralization. After thorough rinsing in 
sterile water or buffer, the demineralized bone matrix 
material is freeze-dried at -72 degrees C for 24 hours. 
35 At this point, the antigen -extracted DFDBA material is 



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saturated with the soluble calcium solution (such as the 
saturated calcium hydroxide solution) and either allowed 
to dry under sterile ambient conditions, or lyophilized 
(freeze-dried) . The calcium enriched DFDBA is then placed 
in a sterile container for storage. 

5 EXAMPLE THIRTY -NINE 

The demineralized bone matrix can be extracted 
with buffers containing lyotropic agents, such as 4 M 
guanidine, 6 M Urea, or 1% sodium dodecyl sulphate, prior 
to treatment with the soluble calcium solution. Protease 

10 inhibitors may also be added to these extracting buffers 
to inhibit degradation of the demineralized bone matrix 
and also the proteins extracted by this process. 
Following extraction of the demineralized bone matrix, the 
bone matrix is rinsed in sterile water and fresh phosphate 

15 buffer. A soluble calcium containing solution, such as a 
saturated soluble solution of calcium hydroxide, is 
applied to the extracted bone matrix, then the treated 
bone matrix is allowed to dry under sterile ambient 
conditions, or by lyophilization. 

20 EXAMPLE FORTY 

Other calcium containing salt solutions may be 
used in this invention. For example, soluble or saturated 
solutions containing calcium acetate, calcium citrate, 
calcium chloride, calcium formate, calcium 

25 glycerophosphate, calcium lactate, calcium laurate, 

calcium oleate, calcium oxide, calcium palmate, calcium 
salicylate, calcium stearate, calcium succinate, or 
calcium sulfate (anhydrous, hemihydrate, dihydrate) would 
be examples of acceptable soluble calcium sources. The 

30 solubility of these compounds in 100 parts of water range 
from as low as 0.003 parts to as high as almost 100 parts. 
Those skilled in the art will realize that other calcium 
containing compounds, in addition to those listed above; 
may be suitable for use in this invention. 



35 



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0 ffY&MPT.E FORTY -ONE 

The calcium or mineral salt modified 
demineralized bone matrix (see Examples Thirty One - 
Thirty Nine) may be added to an organic fibrous or 
nonfibrous material, such as a collagen or gelatin matrix, 
5 in such a manner as to form a enhanced demineralized bone 
matrix- filled porous or semi-porous sponge material. Such 
a sponge may be formed by adding various proportions of 
calcium or mineral salt modified demineralized bone powder 
or particles to either an aqueous or dry powder dispersion 
10 of collagen or gelatin. By way of example, the 

fabrication of such a enhanced- osteogenic sponge, the 
following procedure serves to provide one such possible 
example under the range of possible approaches referred to 
above . 

15 An aqueous collagen dispersion can be produced 

from a purified bovine collagen material by redispersing 
the collagen powder or fleece in an acidic or alkaline 
solution of either dilute hydrochloric acid or sodium 
hydroxide. For example, the dried collagen material can 
20 b e incrementally added to a .01 N solution of HC1 to 
produce anywhere from about a 0.01% to a 5% collagen 
dispersion. The dispersion is mixed thoroughly with a 
Waring Blender under refrigeration with short bursts of 5 
to 10 seconds agitation. The mixed dispersion can be 
25 dialyzed against sterile distilled water at 4 degrees C. 

to reduce the acid concentration while gradually elevating 
the pH of the collagen dispersion to approximately a range 
of from pH 4 to 5.5. The calcium or mineral salt modified 
demineralized bone may then be added incrementally to the 
collagen dispersion at 4 degrees C. Depending on the 
initial pH of the collagen dispersion, various weight 
ratios of demineralized bone may be added to the collagen 
dispersion, ranging anywhere from about 5 weight percent 
of bone matrix to approximately about 95 weight percent 
bone matrix. By way of example, the added demineralized 



30 



35 



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bone may be demineralized bone comprising added calcium or 
mineral salt, partially activated demineralized bone or 
untreated demineralized bone or combinations thereof. For 
example, aqueous collagen/enhanced demineralized bone 
matrix powder dispersion can then be lyophilized into a 
5 sponge and cut into the desired size and configuration 

with a sharp-bladed instrument or a sponge cutting device 
by conventional methods. 

Alternatively, the acidic or alkaline collagen 
dispersion can be lyophilized by conventional methods then 
W may be ground, under cooling with dry ice and/or liquid 

nitrogen, into a powder. This collagen powder can then be 
dry blended with various ratios of enhanced demineralized 
bone matrix powder. After blending, the powder mixture 
can be hydrated with sufficient sterile distilled water to 
15 form a uniform dispersion. This blended collagen/enhanced 
demineralized bone dispersion may then be lyophilized into 
a sponge configuration. The source of the collagen may be 
from a human or animal origin. 

EXAMPLES FORTY -TWO 

20 Demineralized bone matrix powder, which has not 

been surface activated or modified with calcium or mineral 
salts (other than phosphate buffer) , may be added to 
various forms of reconstituted collagen or gelatin as 
described in Example Forty One. The weight ratio of 
' 25 demineralized bone powder to collagen or gelatin matrix 
material is from about 60 weight percent to about 90 
weight percent of the demineralized bone powder matrix 
component. The resultant sponges have the following 
unique properties which enhance their clinical utility: 
3 q i) Enhanced maintenance of shape, form, and 

resilience under moist conditions. 
2) Enhanced resistance to compressibility in 
the dry and/or moist conditions, while 
maintaining an elastic, sponge -like 
35 physical behavior. 



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3) Enhanced space maintaining function, 

4) Enhanced cellular infiltration without a 
significant increase in inflammatory cells, 
such as macrophages. 

If the composition is in the form of a sponge, 
5 preferably it is characterized by a density of about 0.1 
grams/cubic centimeter (cc) or greater than 0.1 
grams/cubic centimeters (cc) . The range of sponge density 
may be from about 0.1 grams/cc to about 0.5 grams/cc, with 
the preferred density from about 0.11 to about 0.35 
10 grams/cubic centimeter. Sponges with about 90 weight 

percent or greater of demineralized bone require a pH for 
matrix collagen (or gelatin) of less than pH 5.0, and 
preferably less than pH 4.5. If the collagen (or gelatin) 
is provided as an acidic powder, for later blending with 
15 the demineralized bone, the pH of the collagen dispersion 
prior to lyophilization and milling into a powder for 
blending, should be less than pH 5.0, and preferably below 
pH 4.5. If the collagen is dispersed in the alkaline 
range, the pH should be above 9.0. The source of the 
20 collagen may be from human or animal origin." 

EXAMPLES FORTY -THREE 
Demineralized bone matrix or reconstituted 
collagen matrix may be treated with concentrations of 
aqueous or soluble alkaline phosphatase ranging from as 
25 low as 10 enzyme units per milligram bone or collagen up 
to or greater than 100 units per milligram bone or 
collagen. Demineralized bone matrix which was determined 
to be inactivate (as tested in the mouse thigh animal 
model) can be converted to active, mineralizing bone by 
30 pre- treatment with 100 units per milligram of alkaline 
phosphatase, followed by dehydration or drying of the 
treated bone. Reconstituted aqueous collagen, containing 
approximately 18 to 20 units of alkaline phosphatase per 
milligram of collagen (dry weight) were lyophilized and 
35 then ground into a fine powder. This collagen- alkaline 



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10 



15 



20 



25 



- 60 - 

phosphatase powder was implanted subcutaneously in mice, 
which resulted in mineralized masses which revealed bone- 
like structures under histologic evaluation. 

While this invention has been described with 
reference to certain specific embodiments, it will be 
appreciated that various modifications of the invention in 
addition to those shown and described herein will become 
apparent to those skilled in the art from the foregoing 
description. Such modifications are included to fall, 
within the scope of the claims appended hereto. 



30 



35 



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What is claimed is: 

.1. An osteogenic composition comprising 
demineralized bone and at least one calcium or other 
mineral containing salt. 

5 2. The composition of claim 1, wherein said 

demineralized bone is partially demineralized bone. 

3. The composition of claim l wherein said 
demineralized bone is demineralized freeze-dried bone 

W allograft. 

4 . The composition of claim 1 wherein said 
demineralized bone in the form of a sponge, particles, 
powder, fleece membrane or fiber. 

15 

5. The composition of claim 5 wherein the 
composition is in the form of a sponge powder, particle, 
fleece, membrane, or fiber. 

20 6. The composition of claim 1, wherein the 

calcium or other mineral containing salt and demineralized 
bone are present at weight proportion or ratio between 
about 0.0001 weight percent to about 20 weight percent. 

25 7. The composition of claim 6 wherein the 

weight proportion or ratio about 0.0010% to about 10%. 

8. The composition of claim 1 in which the 
calcium salt is selected from the group consisting of 
30 calcium acetate, calcium citrate, calcium chloride, 

calcium formate, calcium glycerophophosphate , calcium 
lactate, calcium lacerate, calcium oleate, calcium oxide, 
calcium palistate, calcium salicylate, calcium stearate, 
calcium succinate or calcium sulfate. 



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- 62 - 

9. The composition of claim 9 wherein the salt 
is calcium hydroxide, 

10. The composition of claim 9 wherein the 
calcium hydroxide salt and the demineralized bone are 
present in the composing at the weight proportions or 
ratio is between about 0-001% to about 10% weight. 

11. The composition of claim 1 wherein said 
composition further comprises a material selected from the 
group consisting of vitamins, amino acids, antibiotics, 
bone morphogenetic protein or proteins (BMP) , growth 
factors, see reconstructed collagen, gelatin, fibrin, 
blood proteins or glycerol. 

15 12. A method of making an osteogenic implant 

having enhanced osteogenic potential comprising, sorbing 
at least one soluble calcium or mineral salt into or onto 
demineralized bone. 

20 13 . The method of claim 12 wherein said calcium 

salt is selected from the group consisting of calcium 
acetate, calcium citrate, calcium chloride, calcium 
formate, calcium glycerophophosphate, calcium lactate, 
calcium lacerate, calcium oleate, calcium oxide, calcium 

25 palistate, calcium salicylate, calcium stearate, calcium 
succinate or calcium sulfate. 

14. A method of treating an osseous or 
periodontal defect by applying the composition of claim 1. 



30 



15, A solution of soluble calcium, which when 
applied to a demineralized bone matrix, results in an 
enhancement of the bone formation process. 



35 



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- 63 - 

16. The composition of claim 15 in which the 
calcium solution comprises a solution of calcium 
hydroxide . 

17. The composition of claim 16 in which the 
solution of soluble calcium is rendered sterile. 

18. A method of enhancing bone induction by 
demineralized bone matrix comprising the application of a 
soluble calcium salt to the demineralized bone matrix. 

19 . A method of inducing bone in a hard or soft 
tissue defect comprising the implantation of a composition 
comprising demineralized bone and a soluble calcium salt. 

15 20. An osteogenic composition comprising 

between about 60% to about 95% or about 60% to about 90% 
weight demineralized bone. 

21. The composition of claim 20, wherein said 
20 demineralized bone is partially demineralized bone. 

22. The composition of claim 20 wherein said 
demineralized bone is demineralized freeze-dried bone 
allograft. 

25 23. The composition of claim 20 wherein said 

demineralized bone in the form of a sponge, particles, 
powder, fleece membrane or fiber. 

24. The composition of claim 20 wherein the 
30 composition is in the form of a sponge powder, particle, 

fleece, membrane, or fiber. 

25. The composition of claim 24 wherein said 
composition is in the form of a sponge. 

35 



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26. The composition of claim 25 wherein the 
sponge has a density of about 0.1 grams/cc or greater than 
0.1 grams/cc. 

27. The composition of claim 20 wherein between 
5 about 5% to about 10% to about 40% by weight of the 

composition is comprised of a material selected from the 
group consisting of collagen or gelatin. 



10 



28. The composition of claim 27 wherein the 
collagen is reconstituted collagen. 



29 . An osteogenic composition comprising 
between about 60 percent to 95 percent weight 
demineralized bone and between about 5 percent to 40 

15 percent weight of a material selected from the group 
consisting of collagen or gelatin. 

30. The osteogenic composition of Claim 20 
wherein the composition is comprised of between about 60% 

20 to about 90% demineralized bone. 

31. A method of making an osteogenic 
composition comprising 

25 (a) a dispersing collagen in an acid 

solution having a pH of about 5 or less; 

(b) lyophilizing the acidic collagen 

dispersion; and 

(c) mixing the lyophilized collagen of 
30 step (b) with demineralized bone herein the final 

composition is about 90% by weight demineralized bone. 

32. The composition of claim 31 in the form of 

a sponge. 

35 



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° 33. A composition comprising a carrier and 

alkaline phosphatase, wherein said composition is capable 
of inducing the formation of bone- like mineral structures. 

34. The composition of claim 33 wherein said 

5 carrier is selected from the group consisting of collagen 
and demineralized bone. 

35. The composition of claim 34 wherein said 
alkaline phosphatase is present in a range between about 

10 10 units/milligram carrier to about 5000 units/milligram 
carrier. 

36. The composition of claim 35 wherein the 
range is about 100 units/milligram carrier to about 1000 

15 units/milligram carrier. 

37. The composition of claim 35 wherein said 
composition is in the form of a sponge. 

20 38. A method of treating an osseous or 

peridental defect by applying the composition of claim 20. 

39. A method of treating an osseous or 
peridental defect by applying the composition of claim 27. 

40. A method of treating an osseous or 
peridental defect by applying the composition of claim 33. 

41. An osteogenic composition comprising 95% by 
weight of material selected from the group consisting of: 
(i) untreated demineralized bone; (ii) partially activated 
demineralized bone; (iii) demineralized bone modified by 
the addition of calcium or other mineral containing salt 
or (iv) combinations of (i)-(iii). 

35 



25 



30 



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° 42. An osteogenic composition comprising about 

0 to 95% demineralized bone. 

43. The use of the osteogenic composition of 
claims 1-11 in the manufacture of a medicament for 

5 treating an osseous or periodontal defect in a subject. 

44. The use of the osteogenic composition of 
claims 20-26 in the manufacture of a medicament for 
treating an osseous or periodontal defect in a subject. 



10 



15 



45. The use of the osteogenic composition of 
claims 27-30 in the manufacture of a medicament for 
treating an osseous or periodontal defect in a subject. 

46. The use of the osteogenic composition of 
claims 33-42 in the manufacture of a medicament for 
treating an osseous or periodontal defect in a subject. 



20 



25 



30 



35 



INTERNATIONAL SEARCH REPORT 



A . CLASSIFICATION OF SUBJECT MATTER 



Ins* lional application No. 
Pa/US 96/09749 



IfHLi 6 ^ (IPC) - ~ hnth national clarion and IPC 



B. FIELDS SEARCHED 



, I Minimum documentation searched (classification system followed by classification symbols) 

IPC6: A61K, A61F, A61L 



Documenution searched other than minimum documenution to 



the extent that such documents are included in the fields searched 



Electronic data base 



consulted during the international search (name of data base and. where practicable, search terms used) 



CAPLUS. WPI. EP0D0C 



C DOCUMENTS CONSIDERED TO BE RELEVANT 



Citation of document, with indication, where appropriate, of the relevant passages^ 

Wti, Al, 8904646 (JEFFERIES, STEVEN, R.). 
1 June 1989 (01.06.89) 



US, A, 4472840 (STEVEN R. JEFFERIES), 25 Sept 1984 
(25.09.84), example III, claim 5 



Relevant to claim No. 

1-32,38-39, 
41-45 



33-37,40,42, 
46 



1-32,41, 
43-45 



Further documents are listed in the continuation of Box C [J] See patent family annex. 



Special categories of died documents: 
- document defining the general state of the art which is not considered 
to be of particular relevance 

enier document but published on or after the international filing date 
" document which may throw doubts on priority claim(s) or which is 
' cited to establish the publication date of another citation or other 

special reasoD (as specified) 
)' document referring to an oral disclosure, use, exhibition or other 
I means . 
•P* document published prior to the international filing date but later than 
the pnonty rUte claimed 



T- later document published after the international filing date cr pnonty I 
^ a^n^Snuict with the application but oted to understand 1 
the principle or theory underlying the invention 

'X* document of particular relevance: the claimed invention cannot >be 
roSdard noW cannot be considered to involve an invert 
step when the document is taken alone 

•Y' document of particular relevance: the daimed mvmtion cumot be 
cohered to involve an inventive step when the document « 
Whined with one or more other such documents, such combinanon 
being obvious to a person skilled in the art 

m &T document member of the same patent family 



Date of the aciual completion of the international search 



?fi Sent 1996 

Name and mailing address of the ISA/ 

i European Patent Office, P-B. 5* 1 8 Paicniiaan 2 

JJfl NI.-22S0 HV Rijjwijk 
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iS/' Fax: I - 31 -70) 340-3016 



Date of mailing of the international search report 

1 8. 10. 96 

Authorized officer 
SOFIA NIKOLOP0UL0U 



Form PCT.'ISA,210 (second sheet) (July 1992) 



INTERNATIONAL SEARCH REPORT 



I me 'tional application No. 

PCT/ US 96/ 09749 



Rox 1 Observations where certain claims were found unsearchable (Continuation of hem I of first sheet) 



This international search report has not been established in respect of certain claim, under Article 17(2Xa) for the following reasons: 

a Claims Nos.: 14-19,38-40 ... 

because they relate to subject mater not required to be searched by this Authority, namely: 

Remark • Although these claims are directed to a method of treatment of the 
hTan/anlmal body, the search has been carried out and based on the alleged 
effects of the compound/composition (c.f.PCT Rule 39.H1VJ 

2> n Sclu^y relate to parts of the international application that do not comply with the prescribed requirement, to such 
an extent that no meaningful international search can be earned out, specifically. 



3 ' .□ S^^ey are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4(a). 



Box II Observations where unity of invention is lacking (Continuation of item 2 of first sh eet) 

This International Searching Authority found multiple inventions in this international application, as follows: 



1. □ A« m "quired additional search fees were timely paid by the applicant, this international search report covers all 



searchable claims. 



2. [I A. all searchable claims could be searches without effort justifying an additional fee. this Authority did not invite payment 

— of any additional fee. 



3 PI As only some of the required additional search fee, were timely paid by the applicant, this international search report 
L - 1 covm orty those claims for which fees were paid, specifically claims Nos.: 



4 PI No required additional search fees were timely paid by the applicant C^equenuy this international search report i, 
1 Tewicid to the invention first mentioned in the claims; it .s covered by claims Nos.. 




Form PCT/ISA/210 (continuation of first sheet (l))<July IvM) 



SA ">6767 



INTERNATIONAL SEARCH REPORT 

Information on patent family members 



05/09/96 



International application No. 

PCT/US 96/09749 



Patent document 
cited in search report 



Publication 
date 



Patent family 
- member(s) 



Publication 
date 



W0-A1- 
US-A- 



8904646 
4472840 



01/06/89 
25/09/84 



NONE 



US-A- 



4394370 



19/07/83 



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