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WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




PCX 

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) International Patent Classification ^ ; 
A61L 27/00 



A2 



(11) International Publication Number: WO 99/38543 

(43) International Publication Date: 5 August 1999 (05.08.99) 



(21) International Application Number: PCr/US99/01677 

(22) International Filing Date: 27 January 1999 (27.01.99) 



(30) Priority Data: 
09/014.519 
09/154,400 



28 January 1998 (28.01.98) US 
1 6 September 1 998 ( 1 6.09.98) US 



(71) Applicants {for all designated States except US): REGEN- 

ERATION TECHNOLOGIES, INC. [US/US]; 1 Innova- 
tion Drive, Alachua, FL 32615 (US). UNIVERSITY OF 
FLORIDA TISSUE BANK, INC. [US/USl; 1 Innovation 
Drive, Alachua, FL 32615 (US). 

(72) Inventors; and 

(75) Inventors^AppIicants (for US only): WIRONEN, John, F. 
[US/US]; 1 Innovation Drive, Alachua, FL 32615 (US), 
FELTON, Phillip, A. [US/US]; 1 Innovation Drive, Alachua, 
FL 32615 (US). JAW, Rebecca [US/US]; 1 Innovation 
Drive, Alachua, FL 32615 (US). 

(74) Agent: BENCEN, (jerard, H.; GeranI H. Bencen, P.A., 426 
Anderson Court, Orlando. FL 32801 (US). 



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



Published 

Without international search report and to be republished 
upon receipt of that report. 



(54) Titie: BONE PASTE SUBJECTED TO IRRADIATIVE AND THERMAL TREATMENT 



(57) Abstract 

A thermally sterilized bone paste 
useful in the orthopedic arts, for exam- 
ple in the repair of non-union fractures, 
periodontal ridge augmentation, craniofa- 
cial surgery, implant fixation, impaction 
grafting, or any other procedure in which 
generation of new bone is deemed neces- 
sary, is provided by a composition com- 
prising a substantially bioabsorbable os- 
teogenic compound in a matrix of 11-19 
%. and preferably about 15-19 % (w/w) 
or thermally sterilized gelatin. In var- 
ious embodiments, the osteogenic com- 
pound is selected from (i) demineralized 
bone matrix (DBM); (ii) bioactive glass 
ceramic; BIOGLASS®. bioactive ceramic, 
calcium phosphate ceramic, hydroxyap- 
aiite, hydroxyapatite carbonate, corraline 
hydroxyapatitc, calcined bone, tricalcium 
phosphate, or like material; (iii) bone mar- 
row extracts, vascular proliferation or re- 
generation growth factors, bone morpho- 
genetic protein. TGF-)9, PDGF. or mix- 
tures thereof, natural or recombinant; and 
(iv) mixtures of (i)-(iii). The thermally 
sterilised gelatine may be a commercially 
available grade of gelatine which is both 
thermally and irradiatively sterilized. 



Kinematic Viscosity of Solutions of Porcine and Hunnan 
Gelatin of Dffferen! Heat Treatnnents 




0.0 



T 

0.1 



0.3 0.3 0^ 

Concentration of Solution 



0.6 



121^ hr. Pcnkm (4 samptM) 

130'CO hr. Pofdne (4 MmplM) 

Untrwtad Poiotnt (2 Mmptot) 

DIttIM Watv (2 Mmptet) 

mXm nr. Humm (i sm^) 

Unb«aiadHumwi|Lyo:3xWMH,)I1 Mmpltl 

121'C« hr. Trtrtwl Hum«> (Lyo:3)t WW N,) II »»mpt»l 



FOR THE PURPOSES OF INFORMATION ONLY 



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



AL 


Albania 


ES 


Spain 


LS 


Lesotho 


SI 


Slovenia 


AM 


Armenia 


FI 


Fmland 


LT 


Lithuania 


SK 


Slovakia 


AT 


Austria 


FR 


France 


LI) 


Luxemboufg 


SN 


Senegal 


AU 


Australia 


GA 


Gabon 


LV 


Latvia 


sz 


Swaziland 


AZ 


Azerbaijan 


GB 


United Kingdom 


MC 


Monaco 


TD 


Chad 


BA 


Bosnia and Herzegovina 


G£ 


Georgia 


MD 


Republic of Moldova 


TG 


Togo 


BB 


Barbados 


GH 


Ghana 


MG 


Madagascar 


TJ 


Tajikistan 


BE 


Belgium 


GN 


Guinea 


MK 


The former Yugoslav 


TM 


Turkmenbtan 


BF 


Burlrina Faso 


GR 


Greece 




Republic of Macedonia 


TR 


Turkey 


BG 


Bulgaria 


HU 


Hungary 


ML 


Mali 


TT 


Trinidad and Tobago 


BJ 


Benin 


IE 


Ireland 


MN 


Mongolia 


UA 


Ukraine 


BR 


Brazil 


IL 


Israel 


MR 


Mauritania 


VG 


Uganda 


BY 


Belarus 


IS 


Iceland 


MW 


Malawi 


US 


United States of America 


CA 


Canada 


IT 


Italy 


MX 


Mexicx) 


uz 


Uzbekistan 


CF 


Ccntrar African Republic 


JP 


lapan 


NE 


Niger 


VN 


Viet Nam 


CG 


Congo 


KE 


Kenya 


NL 


Netherlands 


YU 


Yugoslavia 


CH 


Switzeriand 


KG 


Kyrgyzstan 


NO 


Norway 


ZW 


Zimbabwe 


CI 


C6le d'lvoire 


KP 


Democratic Ptoople's 


NZ 


New Zealand 






CM 


Cameroon 




Republic of Korea 


PL 


Poland 






CN 


China 


KR 


Republic of Korea 


PT 


Portugal 






cu 


Cuba 


KZ 


Kazakstan 


RO 


Romania 






cz 


Czech Rcpablic 


LC 


Saint Lucia 


RU 


Russian Federation 






DE 


Germany 


U 


Liechtensteht 


SD 


Sudan 






DK 


Dcnmaiic 


uc 


Sri Lanka 


S£ 


Sweden 






EE 


Estonia 


LR 


Liberia 


SG 


Singapore 







wo 99/38543 PCT/US99/01677 



TITLE OF THF INVPNlTinxT 
BONE PAST E SUBJE C TFD T O IRRADI ATIVF AND THERMAL TREATMFNT 

CROSS REFERENCE TO RF LATED APPT irATlONs; 

This application is a continuation-in-pan of related United States Patent Application serial 
number 09/014.519, nied January 28,1998, pending 

Background of the Invention 

1 . Field of the Invention : 

This invention provides an improved, thermally sterilized bone paste, useftil in the production 
of osteogenic, osteoinductive, and/or osteoconductive compositions for use in the field of 
orthopedic medicine to achieve bone fusions, fusion of implants to bone, filling of bone 
defects, or any other applications in which an osteoinductive, osteogenic composition is 
desirable. 

2. Background : 

More than 100.000 bone grafting procedures are performed every year in the United States 
alone. (Cornell). In the majority of reconstruction procedures, the graft material is used as a 
filler between bone particles in the belief that continuous contact between particles of bone 
leads to more rapid and complete healing at the repair site (as well as greater mechanical 
integrity). (Bloebaum). In the cases of bone augmentation and spinal ftision, these bone grafts 
may make up the entire stnicture of the graft, since there are no bone fragments in the area. 
With the possible exception of one product (whose use guidelines do not allow this), all bone 
grafting materials require surgical placement with the requisite incisions. 

Osteogenic bone grafting materials may be separated into two classes, namely those which 
are osteoconductive. and those which are osteoinductive. While the exact definition of these 
terms remains a matter of debate, it can be said that osteoconductive implants "conduct" bone 



wo 99/38543 PCT/US99/0I677 

2 

growth across defects when implanted into osseous tissue. (Einhom). Osteoinductive 
implants, on the other hand, ha^■e the ability to "induce'* cells in the area to generate bone of 
their own accord. (Einhom). These osteoinductive implants will cause the generation of bone 
even when they are implanted into non-osseous tissue (e.g. subcutaneous or intramuscular 
• implantation). (Einhom; Benedict: Strates; Urisi). 

All of the artificially produced bone-grafting materials available today fall in the 
osteoconductive category of grafts. Among these are Bioglass®. Norian®. Collagrafl®, 
corraiine hydroxyapatite, powdered hydroxyapatite. crystalline and amoiphous hydroxyapatite 
(hydroxy] apatite), and a number of other products. All of these implants rely on their 
similarity to natural bone hydroxyapatite. A likely mechanism for bone conduction lies in the 
ability of these materials to enhance diffijsion of trophic factors and cells over their very large 
surface areas and the mechanical support which they provide to growing tissues. Figure 1 
provides a list of relevant properties of selected bone graft materials. 

15 

The other category of bone grafting materials currently available is encompassed by autograft 
or allograft bone. If not too harshly processed, these materials are generally 
osteoinductive.(Yazdi). Since they are tissue transplants, their use imposes certain risks. 
Autografts have been associated with harvest site morbidity in excess of 20%. (Younger). 
20 Frozen or freeze-dried allografts induce some immune response, and if not properly screened, 
can be associated with disease transmission. (Hordin). The last variety of allografts is 
demineralized bone matrix. 



10 



Demineralized Bone Matrix (DBM) was first described by Senn in 1889. (Senn). It was 
25 rediscovered, largely by accident, and thoroughly studied by Urist and Strates in the late 
1960's. (Strates; Urist). It has since become a major product of tissue banks around the world. 
As the name implies, it is bone which has been demineralized by treatment with acid. A 
detailed outline of the process for producing this product is provided in figure 2. 

30 DBM has the ability to induce the formation of bone even in non-osseous tissues within 4 
weeks. (Strates; Urist; Lasa). The standard technique for determining the activit\' of DBM is 
to implant it subcutaneously or intramuscularly. (Nathan). It is believed that the major active 



wo '''38543 PCT/US99/01677 



factor in DBM is one or more bone morphogenetic proteins (BMP), (see U.S. Patent 
4.294.753. herein incorporated by reference). Other grovMh factors, including but not limited 
to TGF-beta, (see U.S. Patent No. 5.422,340. herein incorporated by reference), platelet 
derived growth factor (PDGF), and the like, may be important for this function also. 

5 

Bioglass® is a bone grafting material which is a SiOj, NajO, CaO, P2O5 glass which has the 
ability to produce a bio-active surface layer of hydroxyapatite carbonate within minutes of 
implantation. (Hench). 

1 0 Two problems are associated with the use of DBM or Bioglass. Both of these materials are 
supplied as large particles, and do not always stay in the area into which they are implanted. 
(Scarborough; Frenkel). Also, due to their coarse nature, they are hard to mold and handle 
in the operating room. Accordingly, there is the need for a product which does not allow for 
particle migration, while also being easier to use in the operating environment. 

15 

As noted in table 1, in recent years, several bone-filling surgical pastes have become 
commercially available. These products range from simple mixtures of saline with a sand-like 
powder to a recently released gel, known as GRAFTON®, a glycerol-based, non-cross- 
linkable composition. All of these products are used in orthopedics to repair bone defects. 
20 such as voids, cavities, cracks etc. Such defects may be the result of trauma or may be 
congenital, and the known pastes may be used to patch or fill such defects, or build upon 
existing bony structures. The ultimate goal of such treatments is that the paste will induce 
bone formation to replace the paste while retaining the form created by the surgeon when 
applying the paste. 

25 

Desirably, a bone paste would be osteoconductive (i.e. it conducts bone cells into a region) 
and osteoinductive (i.e. stem cells are induced to differentiate into bone forming cells which 
begin production of new bone). In general, bone pastes known in the art are osteoconductive, 
with only weak osteoinductive effects. Accordingly, such known pastes are inadequate for 
30 filling of large voids and frequently do not effect proper bone formation even in small voids. 
All currently available bone pastes, including those that exhibit some osteoinductive activity, 
are difficult to handle, do not adequately remain at the site of implantation, or both. 



wo 99/38543 PCT/US99/01 677 

4 

Thus, one commercially available product. GRAFTON®, (see U.S. Patent No. 5.484.601 ) is 
a non-cross-linkabie composition of demineralized bone powder suspended in a polyhydroxy 
compound (e.g. glycerol) or esters thereof, optionally including various other ingredients, 
including gelatin. It is considered likely that this material is rapidly washed away from the 
5 implant location as the carrier matrix is glyceroL which is water soluble. 

U.S. Patent Nos. 5.236.456 and 5,405,390 (O'Leary and Pi^wett) outline an "osteogenic-' gel 
composition which is made from demineralized bone matrix (DBM) by treating with 
concentrated acid (3 M HCl) and heating to between 40 and 50°C. The patent briefly 

0 describes mixing the gel with DBM and several other components. However, the method of 
manufacturing the gel composition is such that it produces mostly collagen fibers (i.e. the 
temperature elevation is insufficient to produce gelatin). As a result, the collagen fibers are 
not soluble in neutral solutions. To obtain a gel. the patent specifies that the collagen must 
be dissolved in acid of low pH (e.g. HCI or 1% acetic acid, at a pH of less than 4.0). 

5 However, compositions of low pH are not typically very compatible with biological 
implantations. It is also noted that at column 5. line 20, and column 6. line 15. it is specified 
that the temperature at which the gel solidifies is 0-5°C, which precludes gelation in vivo. 
U.S. Patent No. 4.440.750 (Glowacki and Pharris) outlines a standard enzymatic technique 
for extracting collagen from tissue using Pepsin. A highly refined collagen is obtained from 

0 animal sources, which is then reconstituted prior to fonning the working composition. The 
collagen will not readily cross-link without the addition of other chemicals (e.g. aldehydes, 
chondroitin sulfate), which they do not specify in the composition. There is no mention of 
a set temperature or any reference to cross-linking behavior. 

5 In U.S. Patent Nos. 4,394,370 and 4,472,840, (JefTeries). complexes of reconstituted collagen 
with demineralized bone or solubilized bone morphogenetic protein, optionally cross-linked 
with glutaraldehyde, were reported to be osteogenic when implanted in vivo. The 
reconstituted collagen of these patents is pulverized, lyophilized, microcr>'stal line collagen 
which has been dialyzed to remove the hydrochloric acid used in collagen preparation. 
Accordingly, the composition of those patents does not involve the conversion of collagen to 
gelatin prior to formation of the composition. Hence, the composition would not exhibit <he 
thermal cross-linking behavior of the instant composition. 



wo 99/38543 PCT/US99/01677 

5 

In U.S. Patent No. 4.678.470 (Nashef e/ al.) disclosed a non-resorbable bone-grafting material 
comprising demineralized bone matrix that had been cross-linked by treatment with 
glutaraldehyde, or lilce cross-linking agent, suspended in a gelatinous or semi-solid carrier. 
Given that the demineralized bone of that patent is chemically cross-linked, its bone inductive 
5 properties are considered to be destroyed and the composition essentially forms a structural 
filler or matrix into which recipient bone may grow. 

In WO 89/04646 (Jefferies). a bone repair material having good structural strength was 
disclosed. The material comprised a demineralized bone matrix which had been surface 
1 0 activated by treatment with glutaraldehyde or like cross-linking agent to increase the binding 
thereof to biocompatible matrices. The resulting material has such a rigid structure that, prior 
to implantation into a biological recipient, the material may be machined. 

In U.S. Patent Application Serial No. 08/816,079, a bone paste comprising gelatin and 
1 5 demineralized bone matrix was disclosed. In that disclosure, the gelatin carrier matrix was 
not subjected to dehydrothermal cross-linking, as is the case for the instam ateliopeptide 
collagen carrier matrix. 



The thermally sterilized bone paste of the present invention meets the needs in the art by 
providing a new material that is easy to handle and store, which adheres to the site of 
implantation, depending on the specific embodiment used, displays both osteoconductive and 
osteoinductive activities, it thermally cross-links at a concentration of between about fifteen 
to about nineteen weight percent at 38°C, as compared to the "Bone Paste'" of U.S. 
Application Serial No. 08/816,079, which thermally cross-linked at between about twenty and 
fony-five weight percent, and is substantially bioabsorbable. Further, as compared with the 
"Thermally Sterilized Bone Paste" of U.S. Application Serial No. 09/014,519. the thermally 
sterilized bone paste of this invention is produced by a novel process including gamma 
irradiation, autoclaving. and blending with constituents by a novel process that permits 
commercial grades of gelatin to be processed for human or animal implantation. 

Preferably, the composition of this invention is provided as a gel which contains mineral and 
protein components which have been clinically shown to induce rapid bone ingrovrth. The 



wo 99/38543 PCT/US99/0I677 

6 

composition may be delivered to the surgeon in a pre-loaded syringe, ready for use. 
Preferably, at a first temperature, the gel is easily formable into any shape, and is adhesive. 
Once inside the biological milieu, or at a second lower temperature, the gel desirabl>' hardens 
as a rubbery solid, which does not wash away or migrate from the site of implantation. Upon 
ingrowth of bone, the implant material becomes completely incorporated into the biological 
system. The mode of making and using this composition is set forth in detail below. 



Brief Summary of the Invention 

A thermally sterilized bone paste useful in the orthopaedic arts, for example in the repair of 
non-union fractures, periodontal ridge augmentation, craniofacial surgery, implant fixation, 
arthrodesis of spinal or other joints, including spinal fusion procedures, or any other procedure 
in which generation of new bone is deemed necessar>'. is provided by a composition 
comprising gelatin and additional osteogenic components. The gelatin is preferably thermally 
cross-linked at about 38°C. at a gelatin concentration of between about 11-30%. and 
preferably at between about I5%-19% (w/w). and the osteogenic components are selected 
from: 

(i) demineralized bone, preferably derived from the species into which the thermally 
sterilized bone paste is to be implanted; or 

(ii) bioactive glass ceramic, BIOGLASS®. bioactive ceramic, calcium phosphate ceramic, 
hydroxyapatite, hydroxyapatite carbonate, corraline hydroxyapatite. calcined bone, 
cortical bone chips, cancellous bone chips, tricalcium phosphate, like material, or 
mixtures thereof; or 

(iii) bone morphogenetic protein, osteogenic proteins or peptides (e.g. osteogenin, pi 5. 
CDMP, and the like), TGF-beta, bone marrow extracts, vascular proliferation or 
regeneration growlh factors. PDGF, or mixtures thereof, natural or recombinant; or 

(iv) mixtures of (i)-(iii). 



Where present (ii) or like material is included to enhance the range of manipulable 
characteristics of strength and osleoinduction exhibited by the composition, and may comprise 
between about 0-60%. including about 40%, of the mass on a weight basis of the composition. 



wo 99/38543 



PCT/US99/01677 



Where present, (iii) reduces the need for demineralized bone, which otherwise provides a 
source of osteoinductive factors. 



Demineralized bone has been shown to be highly effective in inducing bone formation. The 
5 gelatin provides a cross-linkable, adhesive and easily manipulated matrix in which the 
osteoconductive and osteoinductive elements of the composition are carried. Other factors, 
such as antibiotics, bone morphogenetic or other proteins, whether derived from natural or 
recombinant sources, wetting agents, glycerol, dextran, carboxymethyi cellulose (CMC), 
growth factors, steroids, non-steroidal anti-inflammatoiy compounds, or combinations thereof 
1 0 or any other material found to add to the desirable properties of the essential composition of 
this invention may be included. 

The composition may be freeze-dried or pre-constituted, and may be provided in a convenient 
dispensing device, such as a pre-loaded syringe. The gel is preferably in a liquid or highly 
1 5 malleable state at temperatures above about 40°C, but sets up as a hard gel at or preferably 
slightly above the body temperature of the organism into which it is implanted (e.g. at 38°C 
in humans). 



Brief Summary nf the Figures 

20 

Figure 1 is a chart of existing bone grafting materials. 
Figure 2 represents a bone demineralization process. 

Figure 3 is a graph of the kinematic viscosity (centistokes) versus concentration (%) for 
gelatin thermally sterilized or not thermally sterilized in a dry state, followed by 
25 dissolution in water and measurement of the kinematic viscosity. 

Detailed Description of the Invention 



Jt will be appreciated by those skilled in the art that the specifics of the composition of this 
30 invention, its method of preparation and use are applicable to such compositions for use in 
any vertebrate species. Nonetheless, because human use is considered likely to be the 



^^^'^^'^^ PCT/US99/0,677 

8 

principal orthopedic application of this new material, the following description concentrates 
on exemplifying this material for human applications. 

The composition of this invention comprises gelatin and additional osteogenic components. 
5 The gelatin is preferably thermally cross-linked at about 38°C, at a gelatin concentration of 
between about 1 1-30%. and preferably at between about 15%- 19% (w/w), and the osteogenic 
components are selected from: 

(i) demineralized bone, preferably derived from the species into which the thermally 
sterilized bone paste is to be implanted; or 
0 (ii) bioactive glass ceramic. BIOGLASS®. bioactive ceramic, calcium phosphate ceramic, 
hydroxyapatite. hydroxyapatite carbonate, corraline hydroxyapatite. calcined bone, 
cortical bone chips, cancellous bone chips, tricalcium phosphate, like material, or 
mixtures thereof; or 

(iii) bone morphogenetic protein, osteogenic proteins or peptides (e.g. osteogenin, p 1 5. 
5 CDMP, and the like), TGF-beta, bone marrow extracts, vascular proliferation or 

regeneration growth factors, PDGF, or mixtures thereof, natural or recombinant; or 

(iv) mixtures of (i)-(iii). 



) 



The composition is fluid at a first temperature (e.g.. above SSX) and becomes thennally 
cross-linked at or just above a second temperature, corresponding to the normal body 
temperature of the organism into which the composition is to be implanted (e.g., at 38°C in 
humans). 

The terms "thermally cross-linked" or "thennally cross-linkable" are used herein to describe 
the property of a composition which contains molecules which, at or below a given 
temperature and concentration, associate in such a fashion as to result in gelation of a solution 
containing these molecules. 

The temi "thennally sterilized" is used herein to indicate that a material has been treated 
under such conditions of temperature as are generally recognized in the art to render a material 
sterile (i.e. devoid of living organisms). For example, the standard procedure of "autoclaving'' 
a material occurs in a sealed chamber into which steam is pumped to such a pressure that the 



PCT/US99/0,677 



temperature within the chamber reaches approximately 12rC. Treatment of iwenij' minutes 
under such conditions is generally recognized as being sufficient to surface sterilize an object, 
with longer periods being required, depending on the volume of an object or liquid, through 
which heat is to be transferred. Other conditions of do' heat (i.e. absent steam) are also 

5 generally acknowledged as producing a sterile environment, as in, for example, approximately 
121-]30°C of dry heat for from about five minutes to about six hours. Once again, 
considerations of time of exposure are required in order to achieve a sterile field. In any 
event, this term as applied to the composition of this invention does not require that the 
material described as "thermally sterilized'" remain in a sterile state. In other words, the 

0 material may be implanted, in which case it would preferably remain sterile, or it may be left 
exposed on a shelf in an open and contaminated state, and yet still have been "thermally 
sterilized". It is the physical characteristics of the thus-treated material (i.e. molecular weight 
and solution behavior, as revealed by the kinematic viscosity), that is critical, rather than the 
state of being sterile or not. According to this disclosure, treatment of a dried gelatin 

5 composition for approximately 5 minutes to about 1 8 hours, and preferably between about 3-6 
hours at between about 121°C to 130°C is considered to come within the meaning of the term 
"thermally sterilized". 

The term "substantially bioabsorbable" is used herein to describe the property of a material 
) which is no longer detectable at the site of implantation or has been remodeled at that site to 
create endogenous tissue after a reasonable period of biological resorbption. such as three 
months to a year later. Accordingly, for example, demineralized bone matrix which has been 
chemically cross-linked with an agent such as glutaraldehyde, is not considered to be 
substantially bioabsorbable. However, demineralized bone matrix itself, gelatin, and bone 
• morphogenetic factors are all considered to be substantially bioabsorbable as they cooperate 
in new bone formation, rather than purely providing structural rigidity or support, without 
being remodeled into new, endogenous tissue. 

The gelatin acts as a carrier phase and has the ability to thermally cross-link over a very small 
temperature range. This thermal cross-linking reaction is largely controlled by physical 
entanglement and hydrogen bonding between chains, and so is dependant on concentration 
and temperature. (Speriing). Additionally, since gelatin has been used extensively in the 



wo 99/38543 PCTAJS99/01 677 

10 

medical market, its in vivo properties are thoroughly studied. (McDonald). The gel-foam 
sponge is the most familiar application of this biopolymer. Studies have indicated that 
gelatin is only mildly antigenic upon implantation, and is comparable in some of its properties 
to collagen, (McDonald). However, collagen does not exhibit the thermal cross-linking 
5 property so important to the composition of this invention. 

Where present, the bioaclive glass, such as BIOGLASS®, bioactive ceramic, calcium 
phosphate ceramic, hydroxyapatite, hydroxyapatite carbonate, calcined bone, tricalcium 
phosphate, or like material, is included to enhance the range of manipulable characteristics 
0 of strength and osteogenesis (osteoinduction and osteoconduction) exhibited by the 
composition. 



The manufacture of gelatin is based on the partial hydrolysis of collagen. Collagen is 
available from skin, bone, cartilage, tendon and other connective tissue. Skin and bone yield 
Type 1 and Type III collagen molecules, while tendon yields nearly pure Type I collagen, and 
cartilage yields a mixture of Type II and rarer types of collagen molecules. Gelatin molecules 
resemble collagen triple helices, however, they are partially hydrolyzed. As a result, in 
solution they have little organization. But. as the solution cools, the gelatin molecules begin 
to form helical structures. As the solution cools further, the viscosity increases and a phase 
transformation from a solution to a gel occurs. This phase change is reversible when heat is 
added. 



The set time and set temperature of a gelatin solution are dependem on the concentration of 
gelatin in solution, the molecular weight, or intrinsic viscosity, of the gelatin molecules, and 
the pH of the solution. At the isoelectric point, or the pH at which the gelatin molecules are 
electrically neutral, the set time is the shortest. 

Collagen can be partially hydrolyzed by several methods. The Type A process is the simplest 
and most rapid process, in which dilute acid (e.g. less than 1 M HCI) is used to partially 
hydrolyze the collagen. Type A processing is generally used with porcine skin and 
demineralized bovine bone. The Type B process uses an alkaline solution to partially 
hydrolyze the collagen. Type B processing is generally used with bovine hide and 



"^^'^'^^^ PCT/US99/01677 

11 

demineralized bovine bone. Finally. enzymes. such as pepsin, may be used to partially 
hydrolyze collagen. Pepsin preferentially cleaves peptide bonds between aromatic amino 
acids. In collagen, treatment with pepsin converts native collagen, which contains telopeptide, 
to atelopeptide material, which reduces the level of interchain disulfide bonding that can occur 
> in the collagen tertiary structure. 



10 



15 



20 



As one example of this method, the gelatin is prepared from the bones of the species into 
which the compositions are to be implanted, by crushing and defaning the bones followed by 
demineralization in 0.5 N HCl and then soaking for about 24 hours in approximately 300 
mg/L pepsin in a 0.5 M acetic acid at 33°C. We have discovered that the yield of gelatin is 
enhanced by conducting two such extractions, one at about 30°C, and a second at about 33°C, 
and then pooling the product. The pH of the resulting solution is brought to about 7.0 with 
sodium hydroxide to denature the pepsin. The temperature of the solution is raised to between 
about 60-65°C for about 12 to 30 minutes and returned to 19-20°C, or like temperature at 
which the gelatin remains soluble, to effect denaturation of remaining collagen and complete 
conversion to gelatin. The resulting solution is filtered to remove particulates and dialyzed 
or diafiltered against distilled water for 48 hours in a 30K-100K molecular weight cut-off 
(30K-100K MWCO) dialysis or diafiltration membrane. 



In one embodiment of this invention, the gelatin thus produced is next lyophilized, preferably 
in scalable vials. The vials are filled (i.e. the vacuum is replaced) with a diy. inert gas, such 
as nitrogen, argon or the like, sealed, and then autoclaved (or otherwise heated, for example 
in a dry oven, to about 121-130°C), in the sealed environment. This treatment has been 
found to increase the kinematic viscosity of the gelatin upon subsequent solubilization. 
25 permitting a lower effective concentration of the gelatin to be used in the bone paste to 
achieve gelation at about thirty-eight degrees centigrade, than has heretofore been possible. 
Depending on the level of moisture permitted to remain in the sealed environment, the level 
of increase in kinematic viscosity, measured subsequently, can be controlled in a dose- 
dependent fashion (the increase in viscosity is quenched by humidity). After lyophilization 
30 and thermal treatment, the gelatin is redissolved in phosphate buffered saline (PBS) or water 
to a sufficiently high effective concentration such that a final composition comprising from 
about 1 1 -] 9%, and preferably about 1 5-1 9% (w/w) gelatin may be produced. The improved 



^0 99/38S« PCT/US99/01677 

12 

result of this process is that the thus-treated gelatin sets-up as a gel at the effective gelatin 
concentration of about 15-19 weight percent, as opposed to 20-45 weight percent without such 
treatment. This distinction is clearly evident from the differences between the kinematic 
viscosity of gelatin produced as described above, with and without thermal treatment of the 
5 lyophilized material. The viscosity is higher and set temperature lower for the thermall) 
treated material for the same concentration of gelatin when subjected to the lyophilization (or 
other form of drying and moisture reduction) and thermal cross-linking, than when such 
treatment is not conducted. 

0 In a further embodimem of this invention, a commercially available grade of gelatin, such as 
porcine gelatin, is utilized in the manufacture of the Bone Paste. Commercial grades of 
porcine gelatin are well known in the production of medicinal capsules and the like, and have 
also been used to produce a product known as GELFOAM®, an insoluble matrix used in 
hemostatic applications. In this aspect of this embodiment, high-quality, 

5 commercially-available porcine gelatin, such as, for example, 250 to 300. including about 275 
bloom commercial grade porcine gelatin available from DynaGel. Inc.. (Plummer St. & 
Wemworth Ave., Calumet City, IL 60409), Vyse Gelatin Company, (5010 North Rose Street, 
Schiller Park, IL 610176). or the like, is purchased, packaged in an appropriately resistant 
packaging or double packaging, and is then sterilized by exposure to an appropriate dose of 

0 sterilizing radiation. For example, exposure to between about 2-3 MRad of Co"", or an 
equivalently sterilizing dose of radiative exposure from another gamma radiation source. 
Following gamma irradiation, the gelatin is autoclaved under standard autoclave conditions 
known in the art. Subsequent to these treatments, the packaging of the sterilized gelatin is 
checked for integrity. 



Prior to further processing, the sterilized gelatin is removed from the sterile packaging, and 
is ground to a particle size of about 1 .5 mm or less, in a sterile grinder or blender. Subsequent 
to such grinding, the gelatin particles are imimately mixed with demineralized bone matrix 
(DBM), and the resistance of the blend to dissolution in an aqueous solvem at a given desired 
set-temperature is confimied. Thus, for example, if the mixture is to be implanted in a human, 
resistance of the blend to dissolution in water or saline heated to approximately thirty-eight 
degrees centigrade is confirmed. If dissolution occurs, more gelatin is added to the 



"^09^8543 PCT/US99/01677 

13 

formulation to ensure that the mixture is a solid at the desired set-temperature. According to 
this aspect of the invention, it is preferred for the DBM to be present at between about 1% to 
about 37%. and preferably about 24-33%. unless bone morphogenetic proteins (BMP s) or 
other osteogenic growth factors are present in the mixture, in which case the DBM 
5 concentration may be lowered. The gelatin is preferably present at between about 1 2-30%. 
depending on the desired set-temperature. 

Subsequent to blending with DBM. if present, the composition is mixed with a sterile 
calcium-phosphate composition, such as hydroxyapatite. or more preferably, with sterilized 
1 0 cortico-cancellous bone chips. Since corticocancellous chips are only about 30-40% solid 
materiah with the remainder of the volume thereof being void space, on a volume basis, a 
composition comprising about 100% (v/v) corticocancellous chips, with the 60-75% (v/v) of 
the composition being comprised of the gelatinyDBM or gelatin^MP or other growth-factor 
blend, to form the final thermally sterilized bone paste composition of this invention. 

15 

Subsequem to blending, the radiatively and thermally sterilized bone paste composition 
described above is subjected to a ftirther consistency check, to ensure i^sistance to dissolution 
of the bone paste composition in aqueous solution at a temperature at or below the desired set- 
temperature. The criticality of this aspect of the invention being that upon implantation, if the 
20 composition were to immediately liquefy at physiological temperature, the effectiveness of 
the composition in inducing osteogenesis is reduced by too-rapid dissolution of the 
composition. In the event that the final composition does not remain solid at the desired set- 
temperature, increased concentrations of the gelatin are added to ensure solid setup of the 
composition at the desired temperature. 

25 

According to the method of producing the composition of this invention, the gelatin may be 
derived from the same or different species than that into which the composition is to be 
implanted. For example, human, porcine, bovine, fish, equine, feline, or canine gelatin is 
derived from collagen sources such as bone, skin, tendons, or cartilage, and may then be 
30 mixed with DBM or other osteogenic (osteoinductive or osteoconductive) materials. As 
noted above, the collagen is converted to gelatin via. liming, acidification or by enzymatic 
extraction, for example by pepsin or like enzymatic tieatment, followed by denaturation b>' 



0 



I 



"^099,38543 PCT/US99/01677 

14 

heal or other means. The gelatin may be derived from tissue by mastication of the tissue, 
followed by an extended treatment capable of breaking cross-links in the long collagen chains. 
In one embodiment, the tissue is ground then soaked for about 24-72 hours at between about 
2-40°C in dilute acid, such as 0.1 normal acetic acid. Preferably, an enzyme such as pepsin 
at a sufficiently high concentration is added. Pepsin concentrations of between about 10- 
20.000 i.u./liter, 100-2,000 i.u./liter. or like concentrations are added to the dilute acid at the 
start of the treatment, with the period of treatment being adjusted according to the enzyme 
concentration used. Solids are removed from the composition, for example by centriftigation. 
and the supernatant material in solution having a molecular weight of about 50.000 daltons 
or higher is retained. This may be achieved by any of a number of methods known in the art 
including, but not limited to. dialyzing the supernatant in a 50,000 dalton molecular weight 
cut-off membrane against several changes of solution, ultrafiltration against a membrane 
having a like molecular weight cut-ofT, (M WCO) or gel permeation chromatography through 
a medium having a 50,000 dalton molecular mass cut-off. It will be recognized by those 
skilled in the art that the higher the MWCO of the gelatin, the lower the yield. Accordingly, 
lower MWCO gelatin preparations, down to abut 1000 dalton MWCO's could be used, 
recognizing that undesirable low molecular weight species might thereby be retained. Once 
again, the inclusion of the lyophilization and thermal cross linking disclosed above increases 
the yield of higher-molecular weight material. 

In one embodimem of the invention, the gelatin solution resulting from the foregoing 
extraction is denatured, for example by heat-treatment to above about 50 to 65°C. The 
denatured protein is then dried and subjected to an inert-gas. thermal cross-linking step 
described above. Thereafter, the gelatin may be stored in a dry state, or reconstituted with 
physiologically acceptable solutions and stored in a frozen state or it may be freeze-dried after 
reconstitution or it may be precipitated, for example in a volatile organic solvent, and 
reconstituted in a solution, such as an isotonic saline solution, at a concentration of between 
about 1 5- 1 9% (w/w) gelatin. 



The demineralized bone is preferably in a powdered form, and is preferably composed of 
particles in the size range between about 80-850 pm in diameter. Methods for producing 
demineralized bone powder are known in the art (see for example U.S. Patent No. 5.405,390. 



wo 99/38543 PCT/US99/0I677 

IS 

herein incorporated b>' reference for this purpose), and are not. therefore, elaborated here. 
Demineralized bone powder, extracted by standard techniques, is mixed with the gelatin 
solution prepared as described above, to form a composition comprising about 1 -40% (vv/w) 
demineralized bone powder. Where present, bone morphogenetic proteins (BMP) reduce the 
5 percentage of DBM required in the composition. The BMP is preferably present at a 
concentration of between about 0.0001 to 10 mg/mL 0.001 mg/ml to 4 mg/ml. or like 
concentration, depending on the amount of DBM present (1-40% w/w). 

In certain embodiments of this invention, and for particular orthopaedic applications in which 
1 0 strength of the bond formed by the thermally sterilized bone paste is important, addition of 
a bioactive glass is preferred. When added, the bioactive glass lowers the adhesiveness of the 
composition, but increases the stiffness of the composition upon setting. Accordingly, a 
bioactive glass, such as BIOGLASS® having a diameter of between about 0.5-710 ^m, is 
added to the gel/demineralized bone composition. In addition, a composition comprising 
1 5 between about 0-40% (w/w) of bioactive glass with the gelatin forming about 11-19% (w/w) 
of the composition is also contemplated. 

Compositions prepared as described above are easily extruded from a syringe, particularly 
when the temperature is elevated to above about 40°C, for example by immersion in a water 
20 bath, by limited treatment in a microwave, by placement in a syringe warmer, or any of a 
number ofother methods for heating the container. The extruded gel is resilient, sticky and 
easily formable into any desired shape. In addition, the composition retains its strength and 
is poorly soluble in saline or water once it sets-up. 

25 Accordingly, having generally described the composition of this invention, and taking into 
account the specifics of the exemplary support provided below, the following guidelines for 
the preparation and use of the composition of this invention are provided: 

The gelatin from DBM should be prepared at a temperature between about 30 and 37°C. 
30 While the yield is higher (60%) at 37°C, the quality, based on measured kinematic viscosity, 
is slightly lower than that produced at 30°C. Preferably, the gelatin is produced by limited 
exposure of collagen to an en^e, such as pepsin, or like enzyme. A concentration of pepsin 



wo 99/38543 PCT/US99/01 677 

16 

set at 300 U/L-500 U/L works well, but those skilled in the art will recognize thai a wide 
range of enzyme concentrations could be tested, based on what is disclosed herein. Those 
skilled in the art will recognize that acid or alkaline processing of skin and tendon may be an 
alternative to the pepsin technique. 

5 

The final composition preferably comprises gelatin solution having a viscosity of about 3600 
centipoise or higher at 44°C (when measured in the linear range of a viscosity/sheer rate plot 
- 0.87/s). or a kinematic viscosity of about 0.7 centistokes at 44°C. The concentration of the 
gelatin in the carrier phase (i.e. absent added osteogenic components) is preferably about 15- 

10 19% (w/w). to ensure that gelation at 38°C will occur in a reasonable amount of time. 
Naturally, those skilled in the art will recognize that, depending on the species of the organism 
into which the composition is to be implanted, different temperatures may be required. These 
needs are accommodated by altering the gelatin concentration, increasing the concentration 
if a higher gel temperature is desired, and lowering the concentration if a lower gel 

1 5 temperature is desired. 



The DBM content of the composition is defined herein by the concentration required to obtain 
bone formation similar to that seen with DBM alone. We have found that about 5-40% (w/w) 
DBM in the composition is effective. Anything lower than about 5% seems to do very little 

20 by way of bone formation, unless added BMPs (component iii) are present in the composition, 
in which case the DBM concentration may be substantially reduced or eliminated altogether. 
Naturally, based on this disclosure, ihose skilled in the art will recognize that by addition of 
different concentrations and compositions of bone morphogenetic proteins or other osteogenic 
or osteoinductive factors, the weight percent of DBM in the composition may be manipulated 

25 up or down. In addition, it will be recognized that, depending on the species into which the 
composition is implanted, the DBM weight percent may need to be adjusted up or down. 

We have found in in vivo studies that the compositions with DBM contents from 1 5 to 33% 
all produce calcified tissue. We have found that there is a good correlation between the 
30 amount of DBM in the composition and the level of bone induction, as long as the DBM 
concentration is greater than about 1 9% (w/w). About 38-40% (w/w) is the upper mass limit 



wo 99/38543 PCT/US99/01677 

17 

for DBM. Accordingly. 1-40% (w/w) DBM, and more preferably 5-30% (w/w). 7-33%(w/w) 
or 1 5-25% (w/w) is desirable for this component. 

We have obsen'ed histologically that, subsequent to implantation into an animal, the gelatin 
phase is totally absorbed within about 2 weeks. Additionally, cartilage and mineralized bone 
formed within two weeks, with mature bone being evident by about the fourth week. The 
animals in these studies did not exhibit any gross health problems or any indications of 
irritation, hematoma, soreness, fever, or weight loss during the study. 



10 



The composition according to this invention, whether it comprises gelatin and osteogenic 
componems (i-iv) may act as a canrier for cortical cancellous or cortical and cancellous bone 
chips. Such compositions are useful for filling larger bone voids. In addition, when these 
bone chips are not demineralized, they provide an added spectrum of biological properties not 
exhibited by the gelatin alone or the gelatin plus osteogenic components (i-iv). When present, 
15 it is preferred for such bone chips to be in the size range of about 80 pm to about 1 0 1 



' mm. 



In a further embodiment of this invention, the composition of gelatin and osteogenic 
componems (i-iv) is injection molded, vacuum molded, rotation molded, blow molded, 
extruded or otherwise formed into a solid form. Such forms would desirably take the form 

20 of vertebral disks, acetabular hemispheres, formable inserts for repairing acetabular cup 
defects, tubes, ellipsoid shapes for void filling, and intramedullary plugs, which are useful to 
plug the intramedullary canal of various bones (i.e. the marrow containing portion of the 
bone) to prevent bone cement from entering healthy bone tissue. These fomis are produced, 
for example, by raising the temperature of the composition above its liquefaction temperature 

25 (e.g. about 45°C), and allowing the composition to gel in a mold of appropriate shape. For 
such forms, the gelatin content is preferably made as high as possible to ensure that the form 
remains solid upon grafting into a vertebrate recipient. 

Having described the composition of this invention and the method of its preparation, the 
30 manner of using the composition is next set forth. Methods of use of the composition include 
extrusion, via injection, or molding, either by hand or mechanically, to produce appropriately 
shaped implants, either in vivo or ex vivo, with subsequent implantation into a desired 



WO''/38S43 PCT/US99/01677 

18 

implantation site. The composition may be directly applied to the site of non-union fractures, 
injected between vertebrae that are to be flised. molded into any physiologic shape desired and 
applied in any orthopedic context in which osteoconduction, or osteoinduction is desired. The 
composition may likewise be used to coat allograft, autograft, xenograft, metallic, symhetic 
5 bioabsorbabie or any other t>'pe of implant to enhance the osseoimegration of the implant and 
osteoconduction and osteoinduction around the implant. This use of the bone paste 
composition of this invention is particularly useful for porous implants. 

Having generally described the invention, the following examples are provided to show 
0 specific features and applications of the invention. It should be recognized that this invention 
is in no way limited to the specifics of the examples as set forth below, and that the limits of 
this invention are defined by the claims which are appended hereto. 

Example 1: 

^ Procedure for Definition of the Set Temner a ture of the Thermally Sterilized Bone Paste: 

In this procedure, the set temperature of the thermally sterilized bone paste is defined. An 
aliquot of the matrix stored in a frozen state was thawed at approximately 45°C and then 
drawn into a sjTinge. The aliquot was then syringed into a tube which is equilibrated at 38°C 
0 for 15 minutes. Another aliquot was syringed into a vial of distilled water equilibrated at 
38°C and allowed to sit for 1 5 minutes. After this period of time, both aliquots of the matrix 
were solid and there was little or no dissolution of the matrix into the distilled water. 

Example 2: 

^ Method for Defining the Concentration o f Gelatin in the Thermallv Sterilized Bone Paste: 

For each composition, a matrix is established as follows: 
Gelatin(g) DBM(g) Water(g) 
0.15 0.33 0.52 

0.17 0.33 0.50 

0.19 0.33 0.48 



■ W0 99/38S43 PCT/US99/01677 

19 

These masses, in a powder form were placed in a plastic bag or like malleable but liquid 
impermeable container prior to addition of the indicated mass of water. The bag was sealed 
and shaken to mix the powders thoroughly. The water was added and the bag resealed. The 
ingredients were kneaded while the bag was submerged in a water bath set at approximately 
5 42-47°C. and then the composition was tested for set-up at 38°C as described in Example 1 . 

Example 3: 

Comparison of the O.steoinductivitv of r.raftnn® ^PBM in glycerol QsteotechV HRM anH 
Thermally Sterilized Rone Paste with Sust^ended DRM i n an Athvmir Rat Intramuscular 
10 Model: 

Known masses of commercially available Grafton® (DBM in glycerol, Osteotech), DBM. 
powdered DBM, and the Themially sterilized bone paste of this invention in which DBM was 
suspended were implanted intramuscularly into athymic nude rats according to the model of 

1 5 Strates and Urist (Urist, C/in. Oriho. Rel Res. 71 :27 1 -278, 1 970). The osteoinductivity of the 
DBM included in the thermally sterilized bone paste of this invention and that included in 
Grafton® were found to be identical in a standard osteosarcoma induction assay. After 21 
days, explants were removed and analyzed by X-ray and atomic absorption. This analysis 
revealed a calcium deposition yield of: 0.40+ 0.17 g/ml implant for the thermally sterilized 

20 bone paste/DBM composition: 0.039 ± 0.094 g/ml for Grafton®; and 0.15 + 0.072 g/ml for 
DBM alone. Therefore, DBM in the composition of this invention yielded a 2.7 fold increase 
in bone induction than DBM alone, and 10.3 fold the bone induction attributable to 
Grafton®. All of these differences were statistically significant. In addition, the 
osteoinductive effect of Grafton® was variable, forming bone in only six often implants. 

25 The DBM/thermally sterilized bone paste of this invention induced bone formation in all 
implants. 



30 



wo 99/38543 PCT/US99/01677 

20 

Example 4: 

Thermally St erilize d B o ne P aste Production. Kinematic Viscosity, and Critical Concentration 
for Gelation at 38 °C! 

5 The kinematic viscosity of porcine gelatin subjected to different heat sterilization treatments 
( 1 2 1 °C, 6 hours; 1 30°C. 3 hours; 1 50°C, 2.5 hours) was compared to the kinematic viscosity 
of porcine gelatin which had not been subjected to heat sterilization. The lyophilized gelatin 
samples treated for 3 hours at 130°C required approximately 40-50 minutes to dissolve in 
sterile water at 55°C. while the lyophilized gelatin samples treated for 6 hours at 12rC 
1 0 required only about 1 0 minutes to dissolve in sterile water. The porcine material treated for 
2.5 hours at 150°C was not soluble. We found that the kinematic viscosity of the thermalh 
treated porcine samples ( 1 21 "C and 1 30°C) increased, as compared with that of the untreated 
porcine samples. A similar study of human material (12 rC, 6 hours), substantially 
reproduced this result. 

15 

By way of background, the thermally sterilized bone paste was produced from gelatin 
extracted from demineralized human cortical bone powder in the size range of 250 - 850 ^m. 
also referred to as demineralized bone matrix powder (DBM), by treatment with 0.5 M acetic 
acid, and pepsin. The DBM was incubated for from 5 to 24 hours at 30 "C. The supernatant 
20 was retained and the solid material was treated with a fresh solution of acetic acid/pepsin at 
33*0 for another 5 to 24 hours. The supematanis were combined, and the pH was adjusted 
to 7.0 with I N NaOH, deactivating the pepsin. The solution was pumped at a controlled rate 
through a tube submerged in a 60°C water bath such that all portions of the solution were 
subjected to the 60°C treatment for a full 15 minutes, then quenched in ice water. The 

■ 25 solution was centriftiged and the supernatant was either dialyzed or diafiltcred against a 
30,000 daltons molecular weight cut off membrane. The retentate was then lyophilized. As 
a control, dry samples were retained without further heat treatment. The remainder of the 
material was autoclaved in sealed vials that were first evacuated to 100 millitorr and then 
back-filled with nitrogen, and sealed. The autoclavine was continued for either six hours at 

30 121 °C. or the samples were treated for three hours at 1 30°C. 



wo 99/38543 PCT/US99/01677 

21 

In figure 3, the results of heat treating four samples of porcine material at 1 2 1 °C for 6 hours 
(solid circles), four samples of porcine material at 130°C for 3 hours (open circles), is 
compared with two samples of untreated porcine material (solid triangles). In addition, one 
sample of human material treated for 6 hours at 12rC (closed square) is compared with one 
5 sample of untreated human material (closed diamond), and a sample of human material 
treated for six hours at 121X, but under an atmosphere of moist (about 1% humidity) 
nitrogen (open diamond). 



The kinematic viscosities of dilute concentrations of the thus treated materials (0.5%, 0.25%, 
1 0 0.125%. 0.0625% in phosphate buffered saline solutions (pH 7.4 at 25*^0), were measured 
with an Ubbelhode viscometer at 44 °C. Tht kinematic viscosities (centistokes) were graphed 
versus concentration, figure 3. The linear regression was extrapolated to zero to determine 
the kinematic viscosity at zero concentration. 

1 5 To determine the set temperatures for various thermally sterilized bone paste compositions, 
gelatin concentrations were varied from 15 w/w% of total composite to 19 w/w% of total 
composite in water. All thermally sterilized bone paste composites tested contained DBM at 
a concentration of 33 w/v^/o of the total composite. Different ambient temperatures were used 
to test whether the thermally sterilized bone paste was solid or liquid, 45°C, 43°C, 4rC, 

20 40°C, SS^'C. and 35.5^C. The Tset temperature was determined both by subsequent lowering 
of the ambient temperature and raising of the ambient temperature. The critical concenu-ation 
of gelatin in a thermally sterilized bone paste composite that was solid at slightly above 
human body temperature, 38°C to 39X, was 15 w/w% -19 w/w% of the total composite for 
human gelatin, processed at 33°C, and with 33 w/w% of the composite being DBM. the 

15 remainder being PBS or water. The human gelatin processed at 33°C had a zero concentration 
kinematic viscosity of 0.65 centistokes. Human gelatin solutions of lower kinematic 
viscosities were found to have critical concentrations in excess of about 1 9 w/w%. 
Correspondingly, gelatins wxh viscosities higher than about 0.65 centistokes are expected to 
thermally cross-link at concentrations lower than about 15% (w/w). 



30 



wo 99/38543 PCT/US99/01677 

22 

Example 5 

Procedure for the Production of the Thermally sterilized hone paste of this Invention : 

This example provides one procedure for the manufacture of bone paste from gelatin and 
5 demineralized bone. As fractions of the total mass of composition desired, the following 
components are weighed (percentages given are of total composite weight): 
Dry demineralized bone: 1 -40% (w/w) 

Lyophilized thermally 

cross-linkable gelatin: ] ] -30% (w/w) 

1 0 BIOGLASS®: 0-40% (w/w) 

bone morphogenetic protein: 0-10 mg/ml 

These components are thoroughly blended while dry, and the balance of the composition mass 
is made up by addition of water, phosphate buffered saline, or any other physiologically 
1 5 acceptable liquid carrier. The composition may be packaged in this form or lyophilized for 
later reconstruction with water. The malleable properties of the composition are achieved by 
heating the composition to a temperature sufficient to exceed the liquefaction point of the 
gelatin, and then allowing the composition to cool to the temperature at which it gels. 

20 Example 6 

Procedure for Producinu a Radiat ivelv and Thermally Sterilized Bone Paste Composition of 
this Invention: 



According to this aspect of the invention, a commercially available grade of porcine gelatin 
25 was utilized in the manufacture of the Bone Paste. Porcine gelatin. 275 bloom commercial 
grade from DynaGel, Inc., (Plummer St. & Wentworth Ave., Calumet City, IL 60409). was 
purchased, packaged in a sealed double packaging, and sterilized by exposure to 2-3 MRad 
of Co^. Following gamma irradiation, the gelatin was autoclaved under standard autoclave 
conditions (20 minutes. 121 degrees). Subsequent to these treatments, the packaging of the 
30 sterilized gelatin was checked for integrity. 



0 



wo 99/38543 PCT/US99/01677 

23 

Prior to further processing, the sterilized gelatin was removed from the sterile packaging, and 
was ground to a panicle size of about 1.5 mm or less, in a sterile grinder or blender. 
Subsequent to such grinding, the gelatin panicles were intimately mixed with demineralized 
bone matrix (DBM) in a sterile sausage-grinder, and the resistance of the blend to dissolution 
in an aqueous solvent at 38 degrees centigrade. Compositions containing DBM at 
concentrations of about 1% to about 37%, including concentrations of about 24-33%. were 
prepared in this manner. In compositions in which bone moiphogenetic proteins (BMP's) or 
other osteogenic growth factors were added the DBM concentration was reduced to as low as 
0%. The gelatin was included in the composition at concentrations between about 12-30%. 
depending on the desired set-temperature. 

Subsequent to blending with DBM, if present, the composition was mixed with a sterile 
calcium-phosphate composition, such as hydroxyapatite, or with sterilized conico-cancellous 
bone chips. Since corticocancellous chips are only about 30-40% solid material, with the 
remainder of the volume thereof being void space, on a volume basis, a compositions were 
prepared comprising about 1 00% (v/v) conicocancellous chips, with the 60-75% (v/v) of the 
composition being comprised of the gelatin/DBM or gelatin/BMP or other growth-factor 
blend, to form the final thermally and irradiatively sterilized bone paste composition of this 
invention. 

Subsequent to blending, the radiatively and thermally sterilized bone pa.ste composition 
described above was subjected to a ftirther consistency checks, to ensure resistance to 
dissolution of the bone paste composition in aqueous solutions at a temperature at or below 
the desired set-temperature. 

As a result of the foregoing processing, a composition having the following characteristics is 
produced: a melt-flow-index (MFI) from a one cubic-centimeter BD (Beckton-Dickinson). 
slip-tip syringe of greater than about 0.0071 9 g/sec, or greater than about 0.03497 g/sec from 
a five cubic centimeter BD slip-tip syringe at 47 + 2 degrees centigiade when 2644 + 1 gram 
of weight is applied to a plunger of a syringe containing said -composition. A funher 
characteristic of such fonnulations is that it does not dissolve within 5 minutes when placed 
in distilled water at 38 + 0.5 degrees centigrade. 



wo 99/381543 



PCT/US99/01677 



24 

Implantation of these compositions are anticipated to result in induction of bone formation 
at the site of implantation. 

References 

5 

ComelL C. Techniques in Orthopaedics 1992, 7. 55-63. 

Bloebaum, R. D. Human Bone Ingrowth and Materials: Bloebaum. R. D., Ed.: Society for 
Biomaterials: Denver, CO, 1 996. 

Einhom, T. A. Enhancement of Bone Repair Using Biomaterials; Einhom. T. A., Ed.: Society 
0 for Biomaterials: Denver, CO, 1996. 

Benedict, J. J. The Role of Carrier Matrices on Bone Induction In Vivo: Benedict. J. J.. Ed.: 
Society for Biomaterials: Denver, CO, 1996. 

Strates. B.; Tiedeman, J. European Journal of Experimental Musculoskeletal Research 1993, 
2,61-67. 

Urist. M. R. Bone Morphogenelic Protein: Urist, M. R., Ed.; W. B. Saunders Co.: 
Philadelphia, 1992, pp 70-83. 

Yazdi, M.; Bemick. S.; Paule, W.; Nimni, M. Clinical Orthopaedics and Related Research 
1991.262,281-285. 

Younger, E.: Chapman, M. Journal of Orthopaedic Trauma 1989. 3. 192-195. 
Hardin. C. K. Otolaringologic Clinics of North America 1994. 27. 91 1-925. 
Senn, N. The American Journal of the Medical Sciences 1889, 98. 2 1 9-243. 
Urist, M. R.; Huo, Y. K.; Brownell, A. G.; Hohl. W. M.; Buyske. J.; Lietze. A.: Tempst, P.; 
Hunkapiller, M.; DeLange, R. J. Procedures of the National Acadamy of Sciences. USA 1984, 
57,371-375. 

Urist, M. R.; Chang. J. J.; Lietze, A.; Huo, Y. K.; Brownell, A. G.: DeLang, R. J. Methods in 
Enzymology 1987, 146, 294-3 13. 

Lasa. C; Hollinger, J.: Droham, W.: MacPhee. M. Plastic and Reconstructive Surgery 1995. 
P<5, 1409-1417. 

Nathan. R.; Bentz, H.: Armstrong. R.: Piez. K.: Smestad, T.: EHingswonh. L.; McPherson. 
J.; Seyedin, S. Journal of Orthopaedic Research 1988. 6. 324-334. 
Hench, L. L.; Andersson, O. H. Bioactive Glasses: Hench. L. L.; Andersson, O. H.. Ed.: 
World Scientific Publishing Co. Pte. Ltd.: Singapore, 1993, pp 41-63. 



- W0 99/38S43 PCT/US99/0I677 

25 

Scarborough. N. Bom Repair Using Allografts: Scarborough. N., Ed.: Society lor 
Biomaierials. 1996. 

Frenkel. S. R.: Moskovich. R.; Spivak. J.; Zhang. Z. H.; Prewett. A. B. Spine 1993. M. \ 
1639. 

5 Sperling, L. H. ImroJuaion to Physical Polymer Science: John Wiley and Sons. Inc.: New 
York, 1992. 

McDonald. T. O.; Britton. B.; Borgmann, A. R.: Robb. C. A. Toxicology 1977, 7. 37-44. 

Culling. C. F. A.; Allison. R. T.; Barr. W. T. Cellular Pathology Technique: 4 ed.; 

Buttenvorths: London, 1985. 
10 U.S. Patent No. 5.481,601 

U.S. Patent No. 5.236,456 

U.S. Patent No. 5.405.390 

U.S. Patent No. 4,440,750 

U.S. Patent No. 4,394,370 
15 U.S. Patent No. 4.472,840 

U.S. Patent No. 4,678,470 

WO 89/04646 



wo 99/38543 



PCT/US99/01677 



26 
Claims 

1 I . An implaniable bone paste composition comprising thermally sterilized gelatin 

2 as a carrier for substantially bioabsorbable osteogenic components for use in a recipient in 

3 need thereof. 

1 2. The bone paste composition of claim 1 when implanted in non-union fractures, 

2 periodontal ridge augmentation, craniofacial surger>', arthrodesis of spinal or other joints, 

3 spinal fusion procedures, and implant fixation. 

1 3. The composition of claim I wherein the gelatin is thermally cross-linkable at 

2 or slightly above the temperature of the organism into which it is to be implanted. 

1 4. The composition of claim 3 wherein said composition gels at about 38°C. 



1 5. The composition of claim 3 wherein said gelatin is present at a concentration 

2 of between about 11-19% (w/w) gelatin as a fraction of the weight of the composition. 



1 




6. The composition of claim 5 wherein the osteogenic component is selected 


2 


from the group consisting of: 


3 


(i) 


demineralized bone, preferably derived from the species into which the thermally 


4 




sterilized bone paste is to be implanted; or 


5 


(ii) 


bioactive glass ceramic, BIOGLASS®. bioactive ceramic, calcium phosphate ceramic. 


6 




hydroxyapatite, hydroxyapatite x:arbonate, corraline hydroxyapatite. calcined bone. 


7 




cortical bone chips, cancellous bone chips, tricalcium phosphate, like material, or 


8 




mixtures thereof; or 


9 


(iii) 


bone morphogenetic protein, osteogenic proteins or peptides and the like. TGF-beta. 


10 




bone marrow extracts, vascular proliferation or regeneration growth factors. PDGF, 


n 




or mixtures thereof, natural or recombinant: or 


12 


(iv) 


mixtures of (i)-(iii). 



wo 99/38543 PCT/US99/0I677 

27 

1 7. The composition of claim 6 wherein the gelatin, the demineralized bone 

2 matrix, or both are derived from the species into which the bone paste is to be implanted. 

1 8. The composition of claim 7 wherein DBM is present at between about 1 -40% 

2 (w/w) of the total composite weight. 

1 9. The composition of claim 8 wherein DBM is present at between about 1 5-33% 

2 (w/w) of the total composite weight. 



1 



10. The composition of claim 6 wherein the bioactive glass is BlOGLASS®. 



1 11. The composition of claim 6 wherein component (ii) is present at between 

2 about 0-60% (w/w) of the total composition mass. 

1 12. The composition of claim 6 comprising antibiotics, bone morphogenetic or 

2 other proteins, whether derived from natural or recombinant sources, wening agents, glycerol. 

3 carboxymethyl cellulose (CMC), growth factors, steroids, non-steroidal anti-inflammatory 

4 compounds, or combinations thereof 



1 

2 



13. The composition of claim 6 comprising between about 0.0001 to 10 mg/ml 
bone morphogenetic protein, natural or recombinant. 



1 



wo 99/38543 



14. 



PCT/US99/01677 

28 

The composition of claim 1 which is a frozen solution or is freeze-dried. 



1 15. The composition of claim 1 wherein the gelatin is human, bovine, porcine, 

2 ovine, fish, equine, feline, canine or mixtures thereof 

1 16. The composition of claim 1 wherein the gelatin is derived from human 

2 collagen sources via enzymatic, acid or alkaline extraction. 



1 



17. The composition of claim 16 wherein said human collagen sources are human 

2 skin, bone, cartilage, tendon, connective tissue, or mixtures thereof. 

1 1 8. The composition of claim 1 7 produced by: 

2 (a) treating the collagen source with pepsin at about 30°C, separating a soluble 

3 supernatant from an insoluble residue, and retaining the soluble supernatant: 

4 (b) treating the insoluble residue with pepsin at about 33°C, separating a soluble 

5 supernatant from an insoluble residue, and retaining the soluble supernatant; 

6 (c) pooling the thus obtained soluble supematants; 

7 (d) heat denaturing the pooled supematants under controlled conditions to produce 

8 gelatin; 

9 (e) removing the moisture from the gelatin to produce dry gelatin; 

1 0 (f) thermally sterilizing the dry gelatin; and 

1 1 (g) mixing a knovwi mass of the dry gelatin with a knovm mass of osteogenic compound 

1 2 such that the dry gelatin is present at a final concentration of about 1 M 9% (w/w). 



1 



19. The composition of claim 1 8 wherein the denaturation is achieved by heating to 



2 at least 



1 20. The composition of claim 19 wherein the gelatin has a molecular wdght of 

2 greater than about 50,000 dallons. 

1 21 . The composition of claim 20 wherein the step of thermally sterilizing the dr\' 

2 gelatin occurs at between about ]2rCto 1 30^C for between about five minutes and IShours. 



wo 99/38543 PCT/US99/01677 

29 

1 22. The composition of claim 1 wherein the osteogenic component is 

2 demineralized bone matrix in a powdered form, and is composed of particles in the size range 

3 between about 80-850 pm in diameter. 

1 23. The composition of claim 22 comprising about 1 -40% (w/w) demineralized bone 

2 matrix powder, provided that if the demineralized bone matrix powder is absent, then a bone 

3 growth factor is present at a concentration of at least 0.0001 mg/ml. 

1 24. The composition of claim 23 wherein said bone growth factor is 

2 morphogenelic protein, TGF-B. osteoinductive factors, osteoconductive factors, or mixtures 

3 thereof, natural or recombinant. 

1 25. The composition of claim 6 wherein the bioactive glass is BIOGLASS® 

2 having a diameter of between about 0.5-710 |im. 

1 26. The composition of claim 1 further comprising cortical cancellous or cortical 

2 and cancellous bone chips. 

1 27. The composition of claim 26 wherein said bone chips are in the size range of 

2 SO^imtolOmm. 

1 28. The composition of claim 1 which is injection molded, vacuum niolded. 

2 rotation molded, blow molded, extmded or otherwise formed into a solid form. 

1 29. The composition of claim 28 wherein said fonm is selected from vertebral 

2 disks, acetabular hemispheres, tubes, ellipsoid, oblong, and "U" shapes for void filling. 

3 intramedullary plug formation, and impaction grafting. 

1 30. A method for inducing bone formation in vivo in a recipient in need thereof 

2 which comprises implanting an effective amount of an implantable bone paste composition 

3 comprising thermally sterilized gelatin as a carrier for substantially bioabsorbable osteogenic 

4 components. 



wo 99/38543 PCT/US99/01 677 

30 

1 31. The method claim 30 which comprises repairing non-union fractures. 

2 achieving periodontal ridge augmentation, conducting craniofacial surgery, securing implants. 

3 arthrodesis of spinal or other joints, spinal fusion procedures, or impaction grafting, which 

4 comprises implanting said composition at the site in vivo in need of such treatment. 

1 32. The method according to claim 31 which comprises extruding said 

2 composition from a syringe at a first temperature at which it remains liquid or highly 

3 malleable, and forming a resilient, sticky and easily formable shape from said composition 

4 as it gels at a second temperature at or slightly above the body temperature of the organism 

5 into which it is implanted. 

1 33. A method for making an implantable graft which comprises preparing 

2 thermally sterilized composition comprising a thermally cross-linkable gelatin carrier and 

3 suspending therein a substantially bioabsorbable osteogenic component. 

1 34. The method of claim 33 wherein said osteogenic component is selected from: 

2 (i) demineralized bone, preferably derived from the species into which the thermally 

3 sterilized bone paste is to be implanted; or 

4 (ii) bioactive glass ceramic, BIOGLASS®. bioactive ceramic, calcium phosphate ceramic, 

5 hydroxyapatite, hydroxyapatite carbonate, corraline hydroxyapatite, calcined bone. 

6 cortical bone chips, cancellous bone chips, tricalcium phosphate, like material, or 

7 mixtures thereof; or 

8 (iii) bone morphogenetic protein, osteogenic proteins or peptides^and the like. TGF-beia. 

9 bone marrow extracts, vascular proliferation or regeneration growth factors. PDGF, 

1 0 or mixtures thereof, natural or recombinant; or 

1 1 (iv) mixtures of (i)-(iii). 

1 35, The method of claim 34 which further comprises injection molding, vacuum 

2 molding, rotation molding, blow molding, extruding or otherwise foming-said composition 

3 into the desired form of a solid graft, and allowing the composition to solidify at a temperature 

4 at which the gelatin becomes thermally cross-linked. 



wo 99/38543 PCT/US99/01677 

31 

1 36. The method of claim 35 wherein said form is selected from venebral disks. 

2 acetabular hemispheres, tubes, ellipsoid, oblong, and "U" shapes for void filling. 

3 intramedullar}' plug formation, and impaction grafting. 

1 37. The method of claim 35 which comprises raising the temperature of the 

2 composition above its liquefaction temperatxire and allowing the composition to gel in a mold 

3 of appropri ate shape . 

1 38. The method of claim 33 wherein the composition is thermally sterilized by 

2 treatment of the diy gelatin at between about 1 2 rc to 1 30°C for between about 5 minutes to 

3 about 18 hours, prior to suspending therein a substantially bioabsorbable osteogenic 

4 component. 

^ 39. The bone paste composition of claim 1 wherein said gelatin is sterilized by 

2 exposure to a sterilizing dose of gamma irradiation. 

1 40. The composition according to claim 39 comprising, on a volume basis, 60- 

2 75% of component (a), and between 0-1 00% on a volume basis, of component (b), which, if 

3 present, because of the substantial void volume thereof, completely absorbs any volume 

4 contribution of component (a), wherein component (a) comprises: 

5 about 1 1-30% (w/w) gelatin, 24-33% (w/w) demineralized bone matrix, with the 

6 balance being made of water or an aqueous solution; 
and wherein component (b) comprises aseptic corticocancellous bone chips. 



7 



1 41 . The composition according to claim 40 wherein <;omponent (a) comprises 

2 between about 15- 1 9% gelatin. 

1 42. The method according to claim 33 comprising exposing said gelatin to a 

2 sterilizing dose of gamma irradiation. 



1 

2 



43. The method according to claim 42 wherein said gelatin is thermally sterilized 
by autoclaving the gelatin. 



wo 99/38543 PCT/US99/01677 

32 

1 44. The method according to claim 43 wherein said gelatin is a commercially 

2 available grade of porcine gelatin. 

1 45. The method according to claim 44 wherein said gelatin has a bloom number 

2 of between about 250 and 300. 

1 46. The composition according to claim 1 having a melt-flow-index (MFI) from 

2 a one cubic-centimeter BD slip-tip syringe of greater than about 0.0071 9 g/sec, or greater than 

3 about 0.03497 g/see from a five cubic centimeter BD slip-tip syringe at 47 ± 2 degrees 

4 centigrade when 2644 ± 1 gram of weight is applied to a plunger of a syringe containing said 

5 composition. 

1 47. The composition according to claim 1 wherein said composition does not 

2 dissolve within 5 minutes when placed in distilled water at 38 ± 0.5 degrees centigrade. 

1 48. A method of enhancing the osteoconductivity, osteoinductivity or 

2 osseointegration activity of an implant which comprises coating said implant with the 

3 composition of claim 1. 



1 



49, 



An implant coated with the composition of claim 1 . 



wo 99/38543 



PCT/US99/01677 



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Figure 3 

Kinematic Viscosity of Solutions of Porcine and Human 
Gelatin of Differeri! Heat Treatments 




0.60 H 1 ( 1 1 1 H 

0.0 0.1 0.2 0.3 0.4 0.5 0.6 



Concentration of Solution (%w/v) 



121"cy6 hr. Porcine (4 samples) 
— O— 130**C/3 hr. Porcine (4 sampies) 
— Untreated Porcine (2 samples) 
—ST- Distaied Water (2 samples) 

121*C/6 hr. Human (1 sample) 

Untreated Human (Lyo: 3x Wet N,) |1 sample] 

-O- 121**C/6 hr. Treated Human (Lyo:3x Wet Nj) |1 sample) 



SUBSTmfnESHEET<RULE 26) 



WORLD TNTTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




PCX 

INTERNATIONAL APPUCATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) International Patait Classification ^ ; 
A61L 27/00 



A3 



(11) International Publication Number: WO 99/38S43 

(43) International Publication Date: 5 August 1999 (05.08.99) 



(21) International Application Number: PCT/US99/0I677 

(22) International FQlng Date: 27 January 1999 (27.01.99) 



(30) Priority Data: 

09/014,519 
09/154.400 



28 January 1 998 (28.0 1 .98) US 
16 September 1998 (16.09.98) US 



(71) Applicants (for all designated States except US): REGEN- 

ERATION TECHNOLOGIES. INC. [US/US]; 1 Innova- 
tion Drive, Alachua. FL 32615 (US). UNIVERSITY OF 
FLORIDA TISSUE BANK. INC. [US/US]; 1 Innovation 
Drive. Alachua. FL 32615 (US). 

(72) Inventors; and 

(75) Inventors/AppUcants (for US only): WIRONEN, John. F. 
[US/US); 1 Innovation Drive. Alachua. FL 32615 (US). 
FELTON. Phillip, A. [US/US]; 1 Innovation Drive. Alachua. 
FL 32615 (US). JAW. Rebecca [US/US]; 1 Innovation 
Drive. Alachua, FL 32615 (US). 



(74) Agent: BENCEN, Gerard, H.; Gerard H. Bencen. PA.. 
Anderson Court, Orlando. FL 32801 (US). 



426 



(81) Designated States: AL. AM. AT. AU, AZ. BA. BB. BG, BR. 
BY. CA. CH, CN. CU, CZ. DE. DK, EE, ES, H. GB. GD, 
GE. GH, GM. HR. HU, ID, IL, IN. IS. JP. KE. KG, KP, 
KR. KZ. LC. LK, LR, LS, LT. LU. LV, MD, MG. MK, 
MN. MW, MX, NO, NZ, PL, PT. RO. RU, SD. SE, SG, 
SI. SK. SL. TJ. TM, TR. TT. UA. UG, US, UZ. VN, YU, 
ZW, ARIPO patent (GH. GM. KE. LS. MW, SD, SZ, UG. 
ZW), Eurasian patent (AM, AZ. BY. KG, KZ. MD. RU, TJ. 
TM), European patent (AT, BE. CH. CY. DE. DK. ES. Fl, 
FR. GB. GR. IE. IT, LU. MC. NL. PT. SE). OAPI patent 
(BF. BJ, CF. CG. CI, CM. GA, GN, GW, 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, 

(88) Date of publication of the international search report: 

23 September 1999 (23.09.99) 



(54) "Htle: BONE PASTE SUBJECTED TO IRRADIATIVE AND THERMAL TREATMENT 



(57) Abstract 



A thermally sterilized bone paste useful in the orthopedic arts, for example in the repair of non-union fractures, periodontal ridge 
augmentation, craniofacial surgery, implant fixation, impaction grafting, or any other procedure in which generation of new bone is deemed 
necessary, is provided by a composition comprising a substantially bioabsorbable osteogenic compound in a matrix of 11-19 %, and 
preferably about 15-19 % (w/w) or thermally sterilized gelatin. In various embodiments, the osteogenic compound is selected from (i) 
demineralized bone matrix (DBM); (ii) bioactivc glass ceramic, BIOGLASS®, bioactive ceramic, calcium phosphate ceramic, 
hydroxyapatite, hydroxyapatite carbonate, corral ine hydroxyapatite, calcined bone, tricalcium phosphate, or like material; (iii) bone marrow 
extracts, vascular proliferation or regeneration growth factors, bone morphogenetic protein, TGF-p, PDGF, or mixtures thereof, natural or 
recombinant; and (iv) mixmres of (i)-(iii). The thermally sterilised gelatine may be a conunercially available grade of gelatine which is both 
thermally and irradiatively sterilized. 



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. 



AL 


Albania 


ES 


Spain 


LS 


Lesotho 


SI 


Slovenia 


AM 


Annenia 


FI 


Fin land 


LT 


Lithuania 


SK 


Slovakia 


AT 


Austria 


FR 


France 


LU 


Luxembourg 


SN 


Senegal 


AU 


Australia 


GA 


Gabon 


LV 


Latvia 


sz 


Swauland 


AZ 


Azerbaijan 


GB 


United Kingdom 


MC 


Monaco 


TD 


Chad 


BA 


Bosnia and Herzegovina 


GE 


Georgia 


MD 


Republic of Moldova 


TG 


Togo 


BB 


Bart»dos 


GH 


Ghana 


MG 


Madagascar 


TJ 


Tajikistan 


BE 


Belgium 


GN 


Guinea 


MK 


The former Yugoslav 


TM 


Turkmenistan 


BF 


Burkina Faso 


GR 


Greece 




Republic of Macedonia 


TR 


"nukey 


BG 


Bulgaria 


HU 


Hungary 


ML 


Mali 


TT 


Trinidad and Tob^o 


BJ 


Benin 


IE 


Ireland 


MN 


Mongolia 


UA 


Ukraine 


BR 


Brazil 


IL 


Israel 


MR 


Mauritania 


UG 


Uganda 


BY 


Belarus 


IS 


Icelarul 


MW 


Malawi 


US 


United States of America 


CA 


Canada 


IT 


Italy 


MX 


Mexico 


UZ 


Uzbekistan 


CF 


Central African Republic 


JP 


J^an 


NE 


Niger 


VN 


Vict Nam 


CG 


Congo 


K£ 


Kenya 


NL 


Netlwrtands 


YU 


Yugoslavia 


CH 


Switzerland 


KG 


Kyrgytstan 


NO 


Norway 


ZW 


Zimbabwe 


a 


Cftie d'lvoire 


KP 


Democratic People's 


NZ 


New Zealand 






CM 


Cameroon 




Republic of Kofrea 


PL 


Poland 






CN 


China 


KR 


Republic of Korea 


PT 


Portugal 






CU 


Cuba 


KZ 


Kazakstan 


RO 


Romania 






CZ 


Czech Republic 


LC 


Saint Lucia 


RU 


Russian Federation 






DE 


Gcimany 


LI 


Liechtenstein 


SD 


Sudan 






DK 


Denmark 


LK 


Sri l^aidca 


SE 


Sweden 






EE 


Estonia 


LR 


Liberia 


SG 


Singiqwrc 







INTERNATIONAL SEARCH REPORT 



Intel 3nal Application No 

PCT/US 99/01677 



A. CLAS9FICATI0N OF SUBJECT HATTER 

IPC 6 A61L27/00 



Acoording to IrHemational Patent Clasailtcation (IRQ or to t>oth national classification and IPC 



B. FIELDS SEARCHED 



Minimum documentation searched (ciasalfication system toUowved by claasiftcation 8ymt)0ls) 

IPC 6 A61L 



Documentation searched other then minimum documentation to the extent that euch documents are tnctudsd in the fields searched 



Electronic data base consulted during the international search (name of data base end. where practical, search temts used) 



C. DOCUMENTS CONSIDERED TO BE RELEVANT 



Category * Citation of document, with indcation. where appropriate, of the relevant passages 



Relevant to daim No. 



P.x 



UO 98 40113 A (GROOMS JAMIE M :WIRONEN 
JOHN F (US); UNIV FLORIDA RES FOUND (US);) 
17 September 1998 (1998-09-17) 
cited in the application 
the whole document 

US 4 191 747 A (SCHEICHER HANS) 
4 March 1980 (1980-03-04) 
column 5, line 3 - column 6, line 17 

WO 96 39203 A (BIOCOLL LAB INC) 
12 December 1996 (1996-12-12) 
page 20, line 5 - line 20; claims 

EP 0 530 804 A (SHAW ROBERT F) 
10 March 1993 (1993-03-10) 
claims; examples 

-/-- 



1-49 



1-49 



1-17 



1-17 



m 



Further documents are listed in the continuation of box C. 



ID 



Patent family members are listed in annex. 



* Special categories of cited documents : 

*A' document defining the general state of the art which s not 
considered to be of particular relevance 

'B' earter document but published on or after the international 
filing date 

■L" document which may throw doubts on priority claim(8) or 
which is cited to establish the publication date of another 
citation or other special reason (as specified) 

"O' document refening to en oral disclosure, use. exhibition or 
other means 

'P' document published prior to the international filing date but 
later than the priority date dainted 



T* later document published after the international filing date 
or priority date and not in conflict with the application but 
cited to understand the principle or theory underiying the 
invention 

'X* document of particular relevance: the claimed invention 
cannot be considered novel or cennot be considered to 
involve an inventive step when the document is taken elone 

"Y* document of particular relevance; the claimed invention 

cannot be considered to involve an inventive step when the 
document is combined with one or more other euch docu- 
ments, such combination being obvious to a person skilled 
in the art. 

"&* document memt)er of the same patent family 



Date of the acUjal completion of the international search 



27 July 1999 



Date of malting of the internetional search report 



05/08/1999 



Name and mailing address of the ISA 

European Patent Office. P.6.5618 Patenitaan 2 
NL • 2280 HV Rijswijk 
Tel. (431-70) 340-2040. Tx. 31 651 epo nl. 
Fax: (431-70) 340-3016 



Authorized officer 



ESPINOSA, M 



Form PCT/ISA/210 (Moond sheet) (July 1982) 



page 1 of 3 



1 



INTERNATIONAL SEARCH REPORT 


Inter artal Application No 

PCT/US 99/01677 


C^Contlnuatlon) DOCUMENTS CONSIDERED TO BE RELEVANT 


Category ' 


Citation of document, with indcation .where appropriate, of the relevant passages 


Relevant to ciaim No. 


X 


EP 0 329 239 A (FORESTI GIANCARLO) 
23 August 1989 (1989-08-23) 
claims 




1-17 


X 


DATABASE UPI 

Section Ch, Week 8911 

Derwent Publications Ltd., London, GB; 

Class D22, AN 89-080728 

XP0021 10466 

& JP 01 032371 A (NITTA GELATIN KK), 
3 February 1989 (1989-02-03) 
abstract 




1,2 


A 


US 5 405 390 A (0 LEARY ROBERT K ET AL) 
11 April 1995 (1995-04-11) 
cited in the application 
column 7, line 63 - column 8, line 4 




1 


A 


US 5 422 340 A (AMMANN ARTHUR J ET AL) 
6 June 1995 (1995-06-06) 
cited in the application 
claims 




1 


A 


DATABASE UPI 

Section Ch, Week 9347 

Derwent Publications Ltd., London, GB; 

Class A96, AN 93-373633 

XP0021 10467 

& JP 05 277174 A (KYOCERA CORP), 
26 October 1993 (1993-10-26) 
abstract 




1 


A 


DATABASE WPI 

Section Ch, Week 9325 

Derwent Publications Ltd., London, GB; 

Class A96, AN 93-199779 

XP0021 10468 

& JP 05 123390 A (KYOCERA CORP), 
21 May 1993 (1993-05-21) 
abstract 




1 


A 


DATABASE WPI 

Section Ch, Week 9001 

Derwent Publications Ltd., London, GB; 

Class A96, AN 90-004589 

XP0021 10469 

& JP 01 288269 A (TOA NENRYO KOGYO KK), 
20 November 1989 (1969-11-20) 
abstract 

-/- 




1 



Foim PCT/ISAAlO (oontlniiatton xA Mcond timi) (Ju^ 1082) 



page 2 of 3 



INTERNATIONAL SEARCH REPORT ime snaiAppiictionNo 

PCT/US 99/01677 


C.(Cominuatlon) DOCUMENTS CONSIDERED TO BE RELEVANT 


Calegoiy' 


Citation ot document, with indication .where appropriate, of the relevant passages 


Relevant to daim No. 


A 


DATABASE WPI 

Section Ch, Week 8835 

Derwent Publications Ltd., London, GB; 

Class D22, AN 88-247568 

XP002110470 

& JP 63 181770 A (NAGASE M), 
26 July 1988 (1988-07-26) 
abstract 


1 



Form PCT/tSA/210toontiiuia[tion of eeoond »hMl) guV 1992) 



page 3 3 



INTERNATIONAL SEARCH REPORT 



I. . national application No. 

PCT/US 99/01677 



Box I Observations where certain claims were found unsearchable (Continuation of Kern 1 of first sheet) 

This International Search Report has not been established in respect of certain ctainis under Article 17(2)(a) for the following reasons: 

1. Claims Nos,: 

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

Remark: Although clalm(s) 30-32 

is(are) directed to a method of treatment of the human/animal 
body, the search has been carried out and based on the alleged 
effects of the compound/composition. 

2. ^] Claims Nos.: 

because they relate to parts of the International Apptication that do not comply with the prescribed requirements to such 
an extent that no meaningful IntemationaJ Search can be carried out, specffically: 



3. I I Claims Nos.: 

because they are dependent daims artd are not drafted in accordance with the second and third sentences of Rule 6.4(a). 

Box il Observations where unity of invention is lacking (Continuation of item 2 of first sheet) 

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



1. I I As all required additional search fees were timely paid by the applicant, this Intemationai Search Report covers all 
I — ' searchable claims. 



2. I I As an searchable claims could be searched without effort justifying an additional fee, this Authority did not invite payment 
of any additional fee. 



3. j I As only some of the required additional search fees were timely paid by the applicant, this Intemationai Search Report 
< — ' covers only those claims for which fees were paid, speciftcally claims Nos.: 



4. Q No required additional search fees were timely paid by the applicant. Consequently, this Intemationai Search Report is 
restricted to the invention first mentioned in the claims; it is covered by claims Nos.: 



Remark on Protest ] [ The additional search fees were accompanied by the applicant's protest. 

I I No protest accompanied the payment of additional search tees. 



Form PCT/ISA/210 (continuation of first sheet (1)) <July 1998) 



INTERNATIONAL SEARCH REPORT 

informatbn on patent f«mlly members 



imef )nal Application No 

PCT/US 99/01677 



Patent document 
cited in seaich report 



Publication 



Patent family 
memt)er(s) 



Publication 
date 



UO 9840113 



17-09-1998 



AU 6552898 A 



US 4191747 A 04-03-1980 



DE 
AT 
AT 
AU 
AU 
BE 
CA 
FR 
GB 
IT 
JP 
SE 
SE 



2657370 A 
370988 B 
885877 A 
517844 B 
3163477 A 
861969 
1095412 
2374040 
1590340 
1088847 B 
53118887 A 
432872 B 
7714045 A 



A 
A 
A 
A 



WO 9639203 A 12-12-1996 



AU 
CA 
CN 
EP 



6107496 A 
2222626 A 
1192700 A 
0851772 A 



EP 0530804 A 10-03-1993 



US 
AU 
AU 
CA 
IL 
JP 
NO 
NZ 
UO 
ZA 



5270300 A 
657888 B 
2541192 
2116859 
102988 
7500741 
940764 
244060 
9304710 
9206729 



A 
A 
A 
T 
A 
A 
A 
A 



29-09-1998 



29- 06-1978 
25-05-1983 

15- 10-1982 

27- 08-1981 
21-06-1979 

16- 06-1978 
10-02-1981 
13-07-1978 

28- 05-1981 
10-06-1985 

17- 10-1978 

30- 04-1984 

18- 06-1978 



24-12-1996 
12-12-1996 
09-09-1998 
08-07-1998 



14-12-1993 
23-03-1995 
05-04-1993 
18-03-1993 
08-02-1998 

26- 01-1995 
29-04-1994 

27- 07-1997 
18-03-1993 
12-03-1993 



EP 0329239 A 23-08-1989 JP 2005960 A 10-01-1990 

US 5292349 A 08-03-1994 



JP 1032371 A 02-02-1989 NONE 



US 5405390 A 11-04-1995 US 5236456 A 17-08-1993 

CA 2041394 A 18-07-1992 

DE 69118505 D 09-05-1996 

DE 69118505 T 28-11-1996 

EP 0495284 A 22-07-1992 

JP 4246359 A 02-09-1993 



US 5422340 A 06-06-1995 



US 


5158934 


A 


27-10-1992 


AT 


153535 


T 


15-06-1997 


AU 


671721 


B 


05-09-1996 


AU 


6026294 


A 


15-08-1994 


CA 


2151486 


A 


21-07-1994 


OE 


69403439 


D 


03-07-1997 


DE 


69403439 


T 


23-10-1997 


DK 


679097 


T 


22-12-1997 


EP 


0679097 


A 


02-11-1995 


ES 


2105641 


T 


16-10-1997 


GR 


3024277 


T 


31-10-1997 


JP 


8505548 


T 


18-06-1996 


UO 


9415«53 


A 


21-07-1994 


US 


5409896 


A 


25-04-1995 



Foim PCT/ISAAIO (poMnt tamay aniwx) (July 1692) 



page 1 of 2 



INTERNATIONAL SEARCH REPORT 

mtormatton on patent family mem be re 



Inte snat Application No 

PCT/US 99/01677 



Patent document 
cited in search report 



Publication 
date 



Patent family 
memt}er(s) 



Publication 
date 



US 5422340 



US 5604204 A 



18-02-1997 



JP 


5277174 


A 


26-10-1993 


NONE 


JP 


5123390 


A 


21-05-1993 


NONE 


JP 


1288269 


A 


20-11-1989 


NONE 


JP 


63181770 


A 


26-07-1988 


NONE 



Forni PCT/ISAfilO (pttent tamOy annex) (July 1692) 



page 2 of 2 



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