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WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




PCX 

INTERNATIONAL APPUCATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) International Patent Classification 6 : 
A61L 27/00 



Al 



(11) International Publication Number: WO 98/40113 

(43) International Publication Date: 17 September 1998 (17.09.98) 



(21) International Application Number: PCT/US98/04904 

(22) International FUing Date: 12 March 1998 (12.03.98) 



(30) Priority Data: 

08/816,079 



13 March 1997 (13.03.97) 



US 



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

SITY OF FLORIDA TISSUE BANK, INC. [US/US]; 1 In- 
novation Drive, Alachua, FL 32615 (US). UNIVERSITY OF 
FLORIDA RESEARCH FOUNDATION, INC. [US/US]; 
223 Grinter Hall, Gainesville, FL 3261 1 (US). 

(72) Inventors; and 

(75) Inventors/Applicants (for US only): WIRONEN, John. F. 
[US/US]; Unit 101, 707 S.W. 75th Street, Gainesville. FL 
32607 (US). GROOMS, Jamie, M. [USAJS]; 5131 N.W. 
76th Lane, Gainesville, FL 32653 (US). 

(74) Agent: BENCEN. Gerard, H.; Gerard H. Bencen, Pj\., 426 
Anderson Court. Orlando, FL 32801 (US). 



(81) Designated States: AL. AU, BA, BB, BG, BR, CA. CN, CU. 
CZ. EE, GE, GW, HU, ID. IL, IS, JP. KP, KR. LC. LK. 
LR, LT, LV, MG, MK, MN. MX, NO, NZ, PL, RO, SG, 
SI, SK, SL, TR. TT, UA, US. UZ, VN. YU. 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, DE, DK, ES. H, 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 evens of the receipt of 
amendments. 



(54)TiUe: BONE PASTE 



0.95 



0.00 



0.85 



0.80 



0.76 - 



0.70 - 



o 


37 


o 


33 •€ 




30 *C 


V 


4-C 



1 1 1 I I — 

0.0 0.1 0.2 0.3 0.4 0.6 

CoficntnttOQ of Otitis (K) 



0.6 



(57) Abstract 



A 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 gelatin matrix. In various embodiments, the osteogenic 
compound is selected from (i) demineralized bone matrix (DBM); (ii) bioactive glass ceramic. BIOGLASS®, bioactivc ceramic, calcium 
phosphate ceramic, hydroxyapatite, hydroxyapatite carbonate, coixaline hydroxyapatitc. calcined bone, tricaJcium phosphate, or like material; 
(iii) bone morphogenetic protein, TGF-j9, PDGF, or mixtures thereof, natural or recombinant; and (iv) mixtures of (i>-(iii). 



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 


Anncnia 


Fl 


Finland 


LT 


Lithuania 


SK 


Slovakia 


AT 


Austria 


FR 


France 


LU 


Luxembourg 


SN 


Soiegal 


AU 


Australia 


GA 


Gabon 


LV 


Latvia 


sz 


Swaziland 


AZ 


Azerbaijan 


GB 


United Kingdom 


MC 


Monaco 


TD 


Chad 


BA 


Bosnia and Herzegovina 


G£ 


Georgia 


MO 


Republic of Moklova 


TG 


Togo 


BB 


Baitados 


GH 


Ghana 


MG 


Madagascar 


TJ 


Tapkistan 


B£ 


Belgium 


GN 


Guinea 


MK 


The former Yugoslav 


TM 


Turkmenistan 


BF 


Burkina Paso 


GR 


Greece 




Republic of Macedonia 


TR 


Turkey 


BG 


Bulgaria 


HU 


Hungary 


ML 


Mali 


TT 


"ninidad and Tobago 


BJ 


Benin 


IE 


Ireland 


MN 


Mongolia 


UA 


Ukraine 


BR 


Brazil 


IL 


Israel 


MR 


Mauritania 


UG 


UgaiKla 


BY 


Belarus 


IS 


Iceland 


MW 


Malawi 


US 


United States of America 


CA 


Canada 


IT 


Italy 


MX 


Mexico 


U2 


Uzbekistan 


CF 


Central African Republic 


JP 


Japan 


NE 


Niger 


VN 


Viet Nam 


CG 


Congo 


KB 


Kenya 


NL 


Netherlands 


YU 


Yugoslavia 


CH 


Switzerland 


KG 


Kyi^gyzstan 


NO 


Norway 


ZW 


Zimbabwe 


CI 


Cttc d'lvoire 


KP 


Democratic People's 


NZ 


New Zealand 






CM 


Cameroon 




Republic of Korea 


PL 


Poland 






CN 


China 


KR 


Republic of Korea 


PT 


Portugal 






cu 


Cuba 


KZ 


Kazakstan 


RO 


R(»nania 






cz 


Czech Republic 


LC 


Saint Lucia 


RU 


Russian Federatk>n 






DE 


Germany 


U 


Liechtenstein 


SD 


Sudan 






DK 


Denmark 


LK 


Sri Lanka 


SE 


Sweden 






EE 


Estonia 


LR 


Liberia 


SG 


Singapore 







wo 98/401 13 PCT/US98/04904 

1 

BONE PASTE 
Background of the Invention 

1. Fiieja of the tov^ptipi^: 
5 This invention relates to a new osteogenic, osteoinductive composition for use in the 

field of orthopedic medicine to achieve bone fusions, fusion of implants to bone, filling of 
bone defects, or any other application in which an osteoinductive, osteogenic composition is 
desirable. 

10 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 

15 mechanical integrity). (Bloebaum). In the cases of bone augmentation and spinal fusion, these 
bone grafts may make up the entire structure 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 

20 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 growth across defects when implanted into osseotis tissxie. (Einhom). 
Osteoinductive implants, on the other hand, have the ability to "induce" cells in the area to 
generate bone of their own accord. (Einhom). These osteoinductive implants will cause the 

25 generation of bone even when they are implanted into non-osseous tissue (e.g. subcutaneous 
or intramuscular implantation). (Einhom; Benedict; Strates; Urist). 

All of the artificially produced bone-grafting materials available today fall in the 
osteoconductive category of grafts. Among these are Bioglass®, Norian®, Collagraft®, 
corraline hydroxy apatite, powdered hydroxyapatite, crystalline and amorphous hydroxyapatite 



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PCT/US98/04904 



2 

(hydroxyl 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 diffusion of trophic factors and cells over their very large 

surface areas and the mechanical support which they provide to growing tissues. Figure 1 
5 provides a list of relevant properties of selected bone graft materials. 

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). 
10 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. 

Demineralized Bone Matrix (DBM) was first described by Senn in 1889. (Senn). It 

was rediscovered, largely by accident, and thoroughly studied by Urist and Strates in the late 
15 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. 

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 activity 
20 of DBM is to implant it subcutaneously or intramuscularly. (Nathan). It is believed that the 

major active factor in DBM is one or more bone morphogenetic proteins (BMP), (see U.S. 

Patent 4,294,753, herein incorporated by reference). Other growth factors, including but not 

limited to TGF-beta, (see U.S. Patent No. 5,422,340, herein incorporated by reference), 

platelet derived growth faaor (PDGF), and the like, may be important for this fimction also. 
25 Bioglass® is a bone grafting material which is a SiOj, NajO, CaO, PjOj glass which 

has the ability to produce a bio-active surface layer of hydroxylapatite carbonate within 

minutes of implantation. (Hench). 

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 



wo 98/40113 



PCTAJS98/04904 



3 

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 panicle migration, while also being easier to use in the operating 
environment. 

5 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, 
such as voids, cavities, cracks etc. Such defects may be the result of trauma or may be 

10 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 stirgeon when 
applying the paste. 

Desirably, a bone paste would be osteoconductive (i.e. it conducts bone cells into a 
15 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 filling of large voids and fi-equently do not effect proper bone formation even 
in small voids. All currently available bone pastes, including those that exhibit some 
20 osteoinductive activity, are difficult to handle, do not adequately remain at 'the site of 
implantation, or both. 

Thus, one commercially available product, GRAFTON®, (see U.S. Patent No. 
5,484,601) is a non-cross-linkable composition of demineralized bone powder suspended in 
a polyhydroxy compound (e.g. glycerol) or esters thereof, optionally including various other 
25 ingredients, including gelatin. It is considered likely that this material is rapidly washed away 
from the 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 Prewett) outline an 
"osteogenic" gel composition which is made fi-om demineralized bone matrix (DBM) by 
treating with concentrated acid (3 M HCl) and heating to between 40 and 50°C. The patent 



wo 98/40113 



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4 

briefly 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 
5 collagen must be dissolved in acid of low pH (e.g. HCl or 1 % acetic acid, at a pH of less than 
4.0). 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 gellation in vivo, 
U.S. Patent No. 4,440,750 (Glowacki and Pharris) outlines a standard enzymatic 

10 technique for extracting collagen from tissue using Pepsin. A highly refined collagen is 
obtained from animal sources, which is then reconstituted prior to forming 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. 

15 In U.S. Patent Nos. 4,394,370 and 4,472,840, (Jefferies), complexes of reconstituted 

collagen with demineralized bone or solubilized bone morphogenetic protein, optionally cross- 
linked with glutaraldehyde, were reported to be osteogenic when unplanted in vivo. The 
reconstituted collagen of these patents is pulverized, lyophilized, microcrystalline collagen 
which has been dialyzed to remove the hydrochloric acid used in collagen preparation. 

20 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 the 
thermal cross-linking behaviour of the instant composition. 

In U.S. Patent No. 4,678,470 (Nashef et al) disclosed a non-resorbable bone-rafting 
material comprising demineralized bone matrix that had been cross-linked by treatment with 

25 glutaraldehyde, or like cross-linking agent, suspended in a gelatinous or semi-solid <:arrier. 
Given that the demineralized bone of that patent is chemically cross-linked, its bone inductive 
properties are considered to be destroyed and the composition essentially forms a structural 
filler or matrix into which recipient bone may grow. 



wo 98/40113 



PCT/US98/049a4 



5 

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 
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 

5 to unplantation into a biological recipient, the material may be machined. 

The bone paste of the present invention meets the needs in the art by providing a 
material that is easy to handle and store, which adheres to the site of implantation, displays 
both osteoconductive and osteoinductive activities, is thermally cross-linkable, and is 
substantially bioabsorbable. Preferably, the composition is provided as a gel which contains 

10 mineral and protein components which have been clinically shown to induce rapid bone 
ingrowth. The 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 
desirably hardens as a rubbery solid, which does not wash away or migrate from the site of 

15 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 Summarv of the Invention 

20 

A 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 necessary, is provided by a composition comprising gelatin 
25 and additional osteogenic components. The gelatin is preferably thermally cross-linkable, and 
the osteogenic components are selected from: 

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



wo 98/40113 



PCTAJS98/04904 



6 

(ii) bioactive glass ceramic, BIOGLASS®, bioactive ceramic, calcium phosphate ceramic, 
hydroxyapatite, hydroxyapatite carbonate, corraline hydroxy apatite, calcined bone, 
tricalcium phosphate, like material, or mixtures thereof; or 

(iii) bone morphogenetic protein, TGF-beta, 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 osteoinduction exhibited by the composition. 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 gelatm 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, carboxymethyl cellulose (CMC), 
growth factors, steroids, non-steroidal anti-inflammatory compounds, or combinations thereof 
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 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). 

Prief gymmary Qf ih^ Fmm 

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



wo 98/40113 



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7 

Figure 3 is a graph of the kinematic viscosity (centistokes) versus concentration (%) 
for human gelatin processed at various temperatures in phosphate buffered saline 
solution (PBS). 

Figure 4A is a photomicrograph of a section of an implant comprising demineralized 
5 bone matrix (DBM) without any carrier after four weeks intramuscularly in a rat. 

Figure 48 is a photomicrograph of a section of an implant conq)rising 33% DBM in 
gelatin (i.e. the paste of this invention) after four weeks intramuscularly in a rat. 

Detailed Des cription of the Invention 

10 It will be appreciated by those skilled in the art that the specifics of the composition 

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

15 The composition of this invention comprises gelatin and additional osteogenic 

components. The gelatin is preferably thermally cross-linkable, and the osteogenic 
components are selected from: 

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

20 (ii) bioactive glass ceramic, BIOGLASS®, bioactive ceramic, calcium phosphate ceramic, 
hydroxyapatite, hydroxyapatite carbonate, corraline hydroxyapatite, calcined bone, 
tricalcium phosphate, like material, or mixtures thereof; or 
(iii) bone morphogenetic protein, TGF-beta, PDGF, or mixtures thereof, natural or 
recombinant; or 

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

The composition is fluid at a first temperature (e.g., above 38''C) and becomes 
thermally cross-linked at or just above a second temperature, corresponding to the ncmnal 



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8 

body temperature of the organism into which the composition is to be implanted (e.g., at 
38*^0 in humans). 

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

The term "substantially bioabsorbable" is used herein to describe the property of a 
material able to cooperate in and become incorporated with new bone formation. 
Accordingly, for example, demineralized bone matrix which has been chemically cross-linked 

10 with an agent such as gluiaraldehyde, is not considered to be substantially bioabsorbable. 
However, demineralized bone matrix itself, bioactive glass or like ceramics, 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. 

The gelatin acts as a carrier phase and has the ability to thermally cross-link over a 

15 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, (Sperling). Additionally, since gelatin has been used 
extensively in the medical market, its in vivo properties are thoroughly studied. (McDonald). 
The gel-foam sponge is the most familiar application of this biopolymer. Studies have 

20 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 property so important to the composition of this invention. 

Where present, the bioactive glass, such as BIOGLASS®, bioactive ceramic, <:alcium 
phosphate ceramic, hydroxy apatite, hydroxy apatite carbonate, calcined bone, triMlcium 

25 phosphate, or like material, is included to enhance the range of manipulable characteristics 
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 



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9 

yield Type I and Type 111 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 
5 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 dependent on the 
concentration of gelatin in solution, the molecular weight, or intrinsic viscosity, of the gelatin 

10 molecules, and the pH of the solution. At the isoelecuic 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 HCl) is used to 
partially hydrolyze the collagen, Type A processing is generally used with porcine skin and 

15 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 
demineralized bovine bone. Finally, enzymes, such as pepsin, may be used to partially 
hydrolyze collagen. Pepsin preferentially cleaves peptide bonds between aromatic amino 
acids. Pepsin also acts as an esterase, but amides of amino acids are not hydrolyzed. 

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 defatting the bones followed 
by soaking for about 24 hours in approximately 300 mg/L pepsin in a 0.5 M acetic acid at 
SS^'C. The pH of the resulting solution is brought to 9.0 with sodium hydroxide to denature 
the pepsin, then it is returned to 7.0 with hydrochloric acid. The temperature of the solution 

25 is raised to 60°C for about 15 to 30 minutes and returned to 4°C to effect denaturation of 
remaining collagen and complete conversion to gelatin. The resulting solution is filtered to 
remove particulates and dialyzed against distilled water for 48 hours in a 50K-1O0K molecular 
weight cut-off (50K-100K MWCO) dialysis membrane. After lyophilization, the gelatin is 



wo 98/40113 



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10 

redissolved in phosphate buffered saline (PBS) or water to an effective concentration of about 
30-45 weight percent of gelatin in solution. 

The gelatin content of the composition is desirably between about 20-45 % (w/w). The 
gelatin may be derived from the same or different species than that into which the composition 
5 is to be implanted. For example, human, porcine, bovine, equine, or canine gelatin is 
derived from collagen sources such as bone, skin, tendons, or cartilage, and may then be 
mixed with DBM or other osteogenic materials. As noted above, the collagen is converted 
to gelatin via, liming, acidification or by enzymatic extraction, for example by pepsin or like 
enzymatic treatment, followed by denaturation by heat or other means. The gelatin may be 

10 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 

15 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 exanq)le by centrifiigation, 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, 

20 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-off, (MWCO) or gel permeation chromatography through a mediimi 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 

25 preparations, down to abut 1000 dalton MWCO's could be used, recognizing that undesirable 
low molecular weight species might thereby be retained. 

The gelatin solution resulting from the foregoing extraction is preferably denatured, 
for example by heat-treatment to above about 50°C. The denatured protein is then stored in 
a frozen state or it may be freeze-dried or precipitated, for example in a volatile organic 



wo 98/40113 PCT/US98/04904 

11 

solvent, and reconstituted in a solution, such as an isotonic saline solution, at a concentration 
of between about 30-45% (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 ^m in diameter. Methods for producing 
5 demineralized bone powder are known in the art (see for example U.S. Patent No. 5,405,390, 
herein incorporated by 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 foim a composition comprising about 0-40% (w/w) 
demineralized bone powder. Where present, bone morphogenetic proteins (BMP) reduce the 

10 percentage of DBM required in the composition. The BMP is preferably present at a 
concentration of between about 0.0001 to 0.1 mg/ml, 0.001 mg/ml to 0.01 mg/ml, or like 
concentration, depending on the amount of DBM present (0-40% w/w). 

In certain embodiments of this invention, and for particular orthopaedic applications 
in which strength of the bond formed by the bone paste is important, addition of a bioactive 

15 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 /an, is 
added to the gel/demineralized bone composition. In addition, a composition comprising 
between about 0-40% (w/w) of bioactive glass with the gelatin forming 20-45% (w/w) of the 

20 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 bath, by limited treatment in a microwave, by placement in a syringe warmer, or 
any of a number of other methods for heating the container. The extruded gel is resilient, 

25 sticky and easily formable into any desired shape. In addition, the composition retains its 
strength and is poorly soluble in saline once it sets-up. 

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: 



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The gelatin from DBM should be prepared at a temperature between about 30 and 
37°C. 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 hydrolysis of collagen with the assistance of an enzyme, such as pepsin, or like 
5 enzyme. A concentration of pepsin set at 300 U/L-500 U/L works well, but those skilled in 
the art will recognize that 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. 

The fmal composition preferably comprises gelatin having a viscosity of about 3600 

10 centipoise 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 30-45% 
(w/w), (approximately 50-60% w/v), to ensure that gelation at 38°C will occur in a 
reasonable amoimt of time. Naturally, those skilled in the art will recognize that, depending 

15 on the species of the organism into which the composition is to be implanted, different 
temperanjres 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 temperature is desired. 

The DBM content of the composition is defined herein by the concentration required 

20 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 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, those skilled in the art will 

25 recognize that by addition of different concentrations and compositions of bone morphogenetic 
proteins or other osteogenic or osteomductive factors, the weight percent of DBM in the 
composition may be manipulated 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. 



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We have found in in vivo studies that the compositions with DBM contents from 15 
to 33% all produce calcified tissue. We have found that there is a good correlation between 
the amount of DBM in the composition and the level of bone induction, as long as the DBM 
concentration is greater than about 19% (w/w). About 38-40% (w/w) is the upper mass limit 
5 for DBM. Accordingly, 0-40% (w/w) DBM, and more preferably 5-30% (w/w), 7- 
33%{w/w) or 15-25% (w/w) is desirable for this component. 

We have observed 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 

10 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. The 
composition according to this invention, whether it comprises gelatin and osteogenic 
components (i-iv) may act as a carrier for cortical, cancellous or cortical and cancellous bone 
chips. Such compositions are useful for filling larger bone voids. In addition, when these 

15 bone chips are not demineralized, they provide an added spectrum of biological properties not 
exhibited by the gelatm alone or the gelatin plus osteogenic conqx)nents (i-iv). When present, 
it is preferred for such bone chips to be in the size range of about 80 /un to about 10 mm. 

In a further embodiment of this invention, the composition of gelatin and osteogenic 
components (i-iv) is injection molded, vacuum molded, rotation molded, blow molded, 

20 extruded or otherwise formed into a solid form. Such forms would desirably take the form 
of vertebral disks, acetabular hemispheres, 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 forms are produced, for example, by raising the temperature of the 

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



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14 

Those skilled in the an will recognize the many orthopedic applications of bone paste 
of this invention. However, by way of illustration rather than limitation, for purposes of 
arthrodesis of the spine, one particularly preferred mode of using this composition would be 
at an early stage of vertebral disk degeneration or subsequent to trauma. Diagnosis of trauma 
5 or degeneration is followed by formation of a small orifice, or a plurality of small orifices in 
the intervertebral cartilage at the site of degeneration. The bone paste is then injected into the 
mtervertebral space to induce arthrodesis. A similar procedure could be used with other 
joints or bone damage. 

Having generally described the invention, the following examples are provided to 
10 show 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. 

JExample I ; 

15 Gelatin Production. Kinematic Viscositv. and Criti cal Concentration for Gelation at 38^C : 
]n this experiment, the source of collagen was from demineralized human cortical bone 
powder in the size range of 250 - 850 ^m. The demineralized bone matrix powder (DBM), 0.5 
M. acetic acid solution, and pepsin were added to a centrifuge tube. The centrifuge tube was 
tumbled for 24 hours at the desired temperature: 4 °C, 30 ''C, 33 °C or 37 °C. The pH was 

20 adjusted to 9.0 then down to 7.0 with 1 N NaOH and IN HCl, respectively, deactivating the 
pepsin. The solution was placed in a 60 °C water bath for 15 minxites, then quenched in ice 
water. The solution was centrifuged and the supernatant was poured into dialysis membrane 
tubing with a 1000 Daltons molecular weight cut off. The supernatant was dialyzed to obtain 
a 1000:1 dilution factor, fi-ozen and lyophilized imtil completely xlry. This experiment was 

25 performed in quintuplicates for each temperature. 

The kinematic viscosities of dilute concentrations of gelatin, 0.0625 w/v%, 0.1 25 w/v%, 
0.25 w/v%, and 0.5 w/v% in phosphate buffered saline solutions (pH 7.4 at 25 °C), were 
measured with an Ubbelhode viscometer at 44 °C. The kinematic viscosities of human gelatin 
processed at 4 °C, 30 °C, 33 °C, and 37 °C, were measured in duplicates, except for 33 °C 



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15 

which was only measured once. The kinematic viscosities (centistokes) were graphed versus 
concentration of human gelatin solution, figure 3. The linear regression was extrapolated to 
zero to determine the kinematic viscosity at zero concentration. The optimum processing 
temperature was determined by the temperature that yielded the highest solution viscosity at 
5 zero concentration, largest slope of the linear regression, greatest yield, and lastly, the gelatin 
that produced a solid bone composite at slightly above human body temperature. 

As the processing temperature increased, the yield of gelatin, normalized for the same 
pepsin to DBM ratio (0.03% (w/v) pepsin/1 g DBM), increased. The kinematic viscosity at zero 
concentration, or y-intercept, followed a reverse trend. As the processing temperatures 

10 increased, the extrapolated kinematic viscosities decreased. Table 1. 

The human gelatin processed at 30 °C had the highest slope on the kinematic viscosity 
versus concentration plot, 0.40 (centistokes/%), followed by the hiunan gelatin processed at 4 
"^C, 0.26 (centistokes/%), the human gelatin processed at 33 °C, 0.21 (centistokes/%), and lastly 
the human gelatin processed at 37 °C, 0.17 (centistokes/%). Table 1. 

15 In order to correlate the kinematic viscosities to molecular weight of gelatin, the 

kinematic viscosities must be translated into intrinsic viscosities. However, the intrinsic 
viscosities were undefined due to the polyelectrolytic nature of gelatin. As a result, a direct 
relationship between viscosity and molecular weight of human gelatin can not be made. 

20 Table 1 . Physical properties of human gelatin and human gelatin in phosphate buffered saline 
solution. Human gelatin was processed at 4 °C, 30 °C, 33 °C, and 37 °C, resulting from 1 g 



of DBM and 0.03 w/v% pepsin solution in 0.5 N acetic acid: 



Human Gelatin 
Processed at 
Various Temp. 


Average 

Yield 
Percent by 
Weight 


Extrapolated 
y-intercept 
(centistokes) 


Slope of Linear 

Regression 
(centistokes/%) 


Value of 
Linear 
Regression 


4 °C 


6% (n=5) 


0.72 (trial 1&2) 


0.26 (trial 1&2) 


0.985 <trial 1&2) 


30 "C 


18%(n=5) 


0.71 (trial 1&2) 


0.40 (trial 1&2) 


0.993 (trial 1&2) 


33 °C 


30% (n=4) 


0.71 (trial 1) 


0.21 (trial 1) 


0.994 (trial 1) 


37 °C 


60%(n=5) 


0.70 (trial 1&2) 


0.17 (trial 1&2) 


0.996 (trial 1&2) 



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The set temperatures for various bone paste compositions were determined. Table 2. 
Human gelatin made from DBM via pepsin at 33 °C, 35 °C, and 37 "^C was used in the bone 
paste compositions. Gelatin concentrations were varied from 19 w/w% of total composite to 
25 w/w% of total composite (corresponding to 40 w/v% to 60 w/v% gelatin in the carrier 
5 matrix) in a pH 7.4 phosphate buffered saline solution (PBS). All bone paste composites tested 
contained DBM at a concentration of 33 w/w% of the total composite. Different ambient 
temperatures were used to test whether the bone paste was solid or liquid, 45 °C, 43 °C, 41 °C, 
40 **C, 38 °C, and 35.5 ^C. The set temperature was determined both by subsequent lowering 
of the ambient temperature and raising of the ambient temperature. 

10 



Table 2. Ambient temj)eratures corresponding to solidified (non-syringe-able) bone paste 
composites. 



Human Gelatin as a 


37 "C Process Temp 


35 "C Process 


33 "C Process 


Percent of Total 




Temp 


Temp 


Composite Weight 








25 w/w% 


< 35.5 "C 


<35.5 "C 


40 "C 


24 w/w% 


<35.5 "C 


<35.5 "C 


<35.5°C 


22 w/w% 


<35.5 "C 


< 35.5 "C 


<35.5 °C 


21 w/w% 


<35.5 "C 


<35.5 "C 


<35.5 °C 


19w/w% 


<35.5 "C 


<35.5 "C 


<35.5 "C 



Accordingly, the critical concentration of gelatin in a bone paste composite that was 
solid at slightly above human body temperature, 38 °C to 39 °C, was 25 w/w% of the total 
composite for human gelatin, processed at 33 °C, and v«th 33 w/w% of the composite being 
25 DBM, the remainder being PBS. The hiiman gelatin processed at 33 °C had a zero 
concentration kinematic viscosity of 0.71 centistokes. Human gelatin solutions of lower 
kinematic viscosities were found to have critical concentrations in excess of about 25 w/w%. 
Correspondingly, gelatins with viscosities higher than about 0.71 -centistokes are expected to 
thermally cross-link at concentrations lower than about 25% (w/w). 



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Example 2: In Vivo Bone Paste Composition and Activity 

This study demonstrates that the bone paste of this invention is osteoinductive. In 
addition, this study demonstrates particle sizes for the DBM component of the composition 
which operate well in promoting new bone growth in an animal into which it is implanted. 

5 The mtra-muscular rat model is the standard model for testing the osteoinductivity of 

demineralized bone and other osteoinductive factors. Strates et al. have used this model for 
many years (Strates). 

As noted in Example 1 above, we determined that for gelation at 38°C, a gelatin 
concentration of 40-60% w/v (30-45% w/w of the solution absent added osteogenic 

10 components) is required. At this concentration, gelatin acting as a carrier matrix thermally 
cross-links at 38°C within approximately 8 minutes, hi this study we addressed the question 
of how much DBM must be present in this fixed 40-60% gelatin carrier matrix to induce bone 
formation which favorably compares with positive controls. We compared 4 different 
compositions of a DBM/Gelatin composite with both positive and negative controls in a rat 

15 intra-muscular model. 

A. Impj^m Prepa]ratjQ][>: 

The femurs, tibiae, and fibulae were harvested from fresh-kilkd (within 24 hours, 
refrigerated at 4°C) Sprague-Dawley rats. The diaphyses were cut from the bones and the 

20 marrow removed from the mid-shaft with a dissecting probe and sterile water wash. 
Mid-shaft segments were then demineralized in 0.6 M. HCl for 24 hours at 4**C with the mass 
ratio of bone to acid maintained at 1/10 or lower. The bone segments were lyophilized and 
then mixed with dry ice and ground in a lab-scale bone mill. DBM powder was sieved and 
the fraction from 125-450 iixa was retained. 

25 A carrier matrix of 50 % (w/v) gelatin was made by heating phosphate buffered saline 

(PBS) to 60°C and then adding powdered porcine gelatin (Sigma, 3(K) bloom) and stirring 
vigorously. Carrier matrix was allowed to age for 15 minutes (to even out the distribution 
of gelatin in solution) and then it was allowed to cool to 50°C. DBM was added to the gelatin 



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18 

solution at this point in the following amounts: 0 (negative control), 15, 19, 24, and 33% w/w 
of the total composite. The composite was blended thoroughly by hand mixing. 

Implants were prepared by ejecting a thread of composite onto a petri dish. These 
threads were cut into short segments (-4 mm.), weighed, and placed into sterile petri dishes. 
5 Positive controls were prepared by pelletizing DBM mixed with PBS in a centrifuge. To 
maintain pellet integrity during the hazards of surgery, these pellets were frozen and 
implanted as such. 

B. Rat Surgery: 

10 Young Sprague-Dawley rats (200-410 g) were anesthetized with 86 mg/kg Ketamine, 

and 13 mg/kg Xylazine administered intramuscularly (in the thigh). A parallel-mid-line 
_ incision was made from the tip of the stemimi to just above the groin. The lateral aspects of 
the rectus abdominus were accessed by blunt dissection to either side of the animal. Three 
short incisions were made in the muscle on each side and the implants inserted, followed by 
15 1 to 2 stitches with Prolenen 3-0 suture (to mark the location and prevent the ejection of the 
implant mass). One positive or one negative control as well as two experimental con^ositions 
were inserted on each side. Implant locations were random except that each rat had one 
positive control on one side and one negative control on the contralateral side. 

Animals were returned to their cages and provided food and water ad-lib. All 
20 members of the study group were kept for 4 weeks except one animal (Rl) which was 
sacrificed after 2 weeks for histology. 

After 4 weeks, animals were sacrificed with an overdose of Nembutal. The rectus 
abdominus was removed by sharp dissection, removing as much tissue as possible. 

25 C. Explant Analysis: 

Each muscle was notched to mark the superior side of the animal and placed into a 
labeled petri dish. The muscle was X-rayed with mammography ^uipment, using 
mammography film (DuPont). Roentgenograms were analyzed using a digital camera 
attached to an Apple LCD equipped with NIH Image 4. 1 software. Images were thresholded 



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to highlight the implant shadow and then the area of the shadow was determined by pixel 
counting. 

Two of each variety of explant were removed from the muscle and fixed in 10% 
buffered formalin. Histological sections were taken and consecutive sections were stained 
5 with H&E and Masson's trichrome stain. These histological samples were examined by a 
qualified pathologist. 

Remaining explants were cut from the muscle tissue and ashed in a muffle fiimace for 
4.5 hours at 700-750°C. Ash weight was determined and normalized to original implant 
weight. Ash was dissolved in LON HCl and analyzed for calcium content by atomic 
10 absorption spectroscopy. 

All analyses were conducted in a blinded manner with decoding done only after 
processing of the data was complete. 

D. Histology: 

15 Two week histology samples of 15% and 19% DBM composites indicated that bone 

formation was occurring, even at this early date. The route of bone formation is not readily 
apparent, but appears to be endochondral. Four week histology samples revealed that mature 
bone was formed at the site of implantation. The quality of bone formed was comparable to 
that of natural bone as shown by the ash and percent calcium analyses. All implants 

20 containing DBM were found to lead to the production of some bone. Those containing 
greater than about 20% DBM yielded the highest quality bone. Figures 4A and 4B provide 
photomicrographs of sections of implants after four weeks in vivo in the rat intramuscular 
model. We foimd that 33% (w/w) DBM in gelatin carrier (figure 4B) according to this 
invention produced as much new bone as pure, 100% DBM (figure 4A). In these figures, the 

25 following structures are evident: 10 is mature bone, as evidenced by red stain uptake from 
Masson's stain; 20 is new cartilage formation, as evidenced by uptake of blue stain from 
Masson's stain and presence of cells; 30 is residual DBM, as evidenced by uptake of blue 
stain and the absence of cells, from which all cartilagenous and bone structures in the muscle 
cross section arose; and 40 is immature bone, as evidenced by light blue staining and the 



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presence of cells. The cells seen are osteoclasts, degrading the newly formed cartilage, and 
osteoblasts laying down new bone. In addition, vascular infiltration in the mature bone is 
evident in the Masson's stained section, from which the black and white prints were made. 

5 E. Compositional Analysis: 

There was no statistically significant difference, using a 2o test, in ash content between 
the negative control, the positive control, or compositions comprising 15% or 19% (w/w) 
DBM. This does not necessarily imply that these compositions do not work (examination of 
the Roen^enograms obviates this conclusion). Rather, it indicates that the sensitivity of the 

10 ash method does not allow the detection of the difference. Examination of the data for the 
24% and 33% composites indicates that they are significantly better than 19%, 15%, and the 
negative controls, and are not significantly different fi"om the (positive) control, see Table 3: 



TABLE 3: 



Composition 
(%DBM) 


' % yield Asb a^g 3&p|ant^^ 


^ iStandard^^^ i 


0 {r control) 


:./■:=,:. ■m:l/^i■:y^:::r■c'i 




15 


5.5 


12.7 (n=6) 


19 


11.9 


12.2 (n=6) 


24 


34.5 


14.9 (n=5) 


33 


30.0 


8.0 (n=4) 


100 {+ control} 


31.9 


8.8 (n=6) 



25 F. Atomic Absorption Spectroscopy: 

The atomic absorption spectroscopy of ashed compositions of DBM/gelatin composites 
yielded the amount of calcium in the samples. The 15% and 19% compositions did not show 
a statistically significant difference from the negative controls. However, it is expected that 
with greater assay sensitivity, positive effects of DBM at concentrations as low as about 7% 



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21 

(w/w) in gelatin carrier would be measurable. The average calcium content produced by 
compositions greater than or equal to 24% appeared to be proportional to the amount of 
DBM, by weight, in the composition: 

TABLE 4: Comparison between the atomic absorption spectroscopy results of ashed samples 
of six different DBM/gelatin composites explanted from rats after 4 weeks in vivo. 







S&idaid Deviation ( p) 


0 { (-) control} 






15 


3.9 


2.4 (n=4) 


19 


7.3 


7.5 (n=4) 


24 


23.1 


8.7 {n=3) 


33 


28.0 


4.4 (n=4) 


100 {(+) 
control} 


81.3 


30.0 (n=5) 



G. X-Ray Digital Analysis: 

Gross examination/comparison of the x-rays reveals that the 24% and 33% 
compositions are not significantly different from the (+) controls. The 15% and 19% 
compositions do not appear to generate significant bone. However, it is expected that with 
greater assay sensitivity, positive effects of DBM at concentrations as low as about 7% (w/w) 
in gelatin carrier would be measurable. No bone formation was apparent on the x-rays at the 
locations of the (-) controls. Accordingly, we conclude that DBM at a concentration of 
between about 24% to 33% (w/w) in gelatin is active in inducing bone formation. These 
same data indicate that concentrations of DBM below about 20% are less effective in 
generating significant bone in comparison to positive controls. It is noted that Grafton™ 
contains only 8% DBM in a glycerol carrier. 



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22 

nw 5: 



Composition 






0 {(-) control) 






15 






19 


4.1 


4.2 (n=7) 


24 


33.0 


15.2 (n = 10) 


33 


36.7 


14.9 (n= 10) 


100 {(+) 
control) 


100 


43.1 (n=10) 



Example 3 

Procedure for the Production Bone Paste of this Invention: 

This example provides one procedure for the manufacture of bone paste from gelatin 
15 and 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: 0-40% (w/w) 
Lyophilized thermally 
20 cross-linkable gelatin: 20-45% (w/w) 
BIOGLASS®: 0-40% (w/w) 

bone morphogenetic protein: 0.001 mg/ml 

These components are thoroughly blended while dry, and the balance of the composition mass 
25 is made up by addition of water, phosphate buffered saline, or any other physiologically 
acceptable liquid carrier. The composition may be packaged in this form or lyophihzed for 
later reconstruction with water. The malleable prq)erties of the con^osition are achieved by 



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heating the composition to a temperature sulficient to exceed the liquefaction point of the 
gelatin, and then allowing the composition to cool to the temperature at which it gels. 

References 

5 

Cornell, 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, 1996. 

Einhom, T. A, Enhancement of Bone Repair Using Biomaterials; Einhom, T. A., Ed.; 
10 Society 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. 

15 Urist, M. R. Bone Morphogenetic 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, 5, 192-195. 
20 Hardin, C. K. Otolaringologic Clinics of North America 1994, 27, 91 1-925. 
Senn, N. The American Journal of the Medical Sciences 1889, 98, 219-243. 
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, 
25 87,371-375. 

Urist, M. R.; Chang, J. J.; Lietze, A.; Huo, Y. K.; Brownell, A. G.; DeLang, R. J. Methods 
in Enzymology 1987, 746, 294-313. 

Lasa, C; HoUinger, J.; Droham, W.; MacPhee, M. Plastic and Reconstructive Surgery 1995, 
96, 1409-1417. 



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24 

Nathan, R.; Bentz, H.; Armstrong, R.; Piez, K.; Smestad, T.; EUingsworth, 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. 
5 Scarborough, N. Bone Repair Using Allografts; Scarborough, N., Ed.; Society for 
Biomaterials, 1996. 

Frenkel, S. R.; Moskovich, R.; Spivak, J.; Zhang, Z. H.; Prewett, A. B. Spine 1993, 18, 
1634-1639. 

Sperling, L. H. Introduction to Physical Polymer Science; John Wiley and Sons, Inc.: New 
10 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.; 

Butterworths: London, 1985. 

U.S. Patent No. 5,481,601 
15 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 

U.S. Patent No. 4,472,840 
20 U.S. Patent No. 4,678,470 

WO 89/04646 



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Claims 



1 1 . An implantable bone paste composition comprising gelatin as a carrier for 

2 substantially bioabsorbable osteogenic components for use in a recipient in need thereof. 

1 2. The bone paste of claim 1 for use in the repair of non-union fractures, 

2 periodontal ridge augmentation, craniofacial surgery, arthrodesis of spinal or other joints, 

3 spinal fusion procedures, and implant fixation. 

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

2 at 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 20-45% (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 matrix (DBM); 

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

5 phosphate ceramic, hydroxy apatite, hydroxy apatite carbonate, corral ine 

6 hydroxyapatite, calcined bone, tricalcium phosphate, or mixtures ther-eof; 

7 (iii) bone morphogenetic protein, TGF-beta, PDGF, or mixtures thereof, natural 

8 or recombinant; and 

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



1 
2 



7. The composition of claim 6 wherein the gelatin, the demineralized bone 
matrix, or both are derived from the species into which the bone paste is to be implanted. 



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26 

1 8. The composition of claim 7 wherein DBM is present at between about 0- 

2 40% (w/w) of the total composite weight. 

1 9. The composition of claim 8 wherein DBM is present at between about 15- 

2 33% (w/w) of the total composite weight. 

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

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

2 about 0-40% (w/w) of the total composition mass. 

1 12. The conq)osition of claim 6 comprising antibiotics, bone morphogenetic or 

2 other proteins, whether derived from natural or recombinant sources, wetting agents, 

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

4 inflammatory compounds, or combinations thereof. 

1 13. The composition of claim 6 conqjrising between about 0.0001 to 0. 1 mg/ml 

2 bone morphogenetic protein. 

1 14. 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, ovine, 

2 equine, 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. 



wo 98/40113 



PCTAJS98/04904 



27 

1 18. The composition of claim 17 produced by treating the collagen source with 

2 pepsin at about 33 °C, heat denaturing the thus treated collagen under controlled conditions 

3 to produce gelatin, and mixing the thus produced gelatin with an osteogenic compound 

4 such that the gelatin is present at a final concentration of about 20-45% (w/w). 

1 19. The composition of claim 18 wherein the denaturation is achieved by heating 

2 to at least 50^*0. 

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

2 greater than about 50,000 daltohs. 

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

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

3 range between about 80-850 /xm in diameter. 

1 22. The composition of claim 21 comprismg about 0-40% (w/w) demineralized 

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

3 then a bone growth factor is present at a concetration of at least 0.0001 mg/ml. 

1 23 . The composition of claim 22 wherein said bone growth factor is morphogenetic 

2 protein, TGF-fi, or mixtures thereof, natural or recombinant. 

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

2 a diameter of between about 0.5-710 /iim. 

1 25. The composition of claim 1 fiirther comprising <;ortical, cancellous or cortical 

2 and cancellous bone chips. 



wo 98/40113 



PCT/US98/04904 



28 

1 26. The composition of claim 25 wherein said bone chips are in the size range of 

2 80|xm to 10 mm. 

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

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

1 28. The composition of claim 27 wherein said form 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 29. 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 gelatin as a carrier for substantially bioabsorbable osteogenic components. 

1 30. The method claim 29 which comprises repairing non-union fractures, achieving 

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

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

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

1 31 . The method according to claim 30 which comprises formation of a series of 

2 small apertures in an intervertebral space and injection of said composition into said space 

3 to induce artherodesis. 

1 32. The method according to claim 30 which comprises extruding said composition 

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

3 maleable, 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. 



wo 98/40113 



PCT/US98/04904 



29 

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

2 composition comprising a thermally cross-linkable gelatin carrier and suspending therein 

3 a substantially bioabsorbable osteogenic component. 

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

2 (i) demineralized bone matrix (DBM); 

3 (ii) bioactive glass ceramic, BIOGLASS®, bioactive ceramic, calcium phosphate 

4 ceramic, hydroxyapatite, hydroxyapatite carbonate, corraline hydroxy apatite, 

5 calcined bone, tricalcium phosphate, or like material; 

6 (iii) bone morphogenetic protein, TGF-fi, PDGF, or mixtures thereof, natural or 

7 recombinant; and 

8 (iv) nuxtures 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 forming said 

3 composition into the desired form of a soUd graft, and allowing the composition to solidify 

4 at a temperature at which the gelatin becomes thermally cross-linked. 

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

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

3 intramedullary plug formation, and infraction grafting. 

1 37. The method of claim 36 which comprises raising the temperature of the 

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

3 mold of appropriate shape. 



wo 98/40113 



PCTAJS98«M904 



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PCTAJS98/04904 



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FIG. 4B 



SUBSTITUTE SHEET (RULE 26) 



INTERNATIONAL SEARCH REPORT 



rnational Application No 

PCT/US 98/04904 



A. CLASSIFICATION OF SUBJECT MATTER 

IPC 6 A61L27/00 



According to tntemational Patent Classification (I PC) or to both national classification and IPC 



B. FIELDS SEARCHED 



Minimum documentation searched (classilicatton system followed by classification symbols) 

IPC 6 A61L 



Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched 



Electronic data base consutted during the international search (name of data base and, where practical, search terms used) 



C. DOCUMENTS CONSIDERED TO BE RELEVANT 



Category ^ 



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



Relevant to claim No. 



wo 96 39203 A (BIOCOLL LAB INC) 12 
December 1996 

see page 20, line 5 - line 20; claims 

EP 0 530 804 A (SHAW ROBERT F) 10 March 
1993 

see claims; example 

EP 0 329 239 A (FOREST! GIANCARLO) 23 
August 1989 
see claims 

DE 42 16 496 A (SATTEL WERNER PROF OR MED 
;ALLPHAMED ARZNEIMITTEL GMBH (DE)) 25 
November 1993 
see claims 

-/-- 



1-37 



1-37 



1,2,6,12 



1,2,6,12 



[ X I ^"'^^r documents are fisted in the continuatton of box C. 



Patent family members are listed in annex. 



" Special categories of cited documents : 

"A" document defining the general state ol the an which Is not 
considered to be of particular relevarKe 

"E" earlier document but published on or after the international 
filing date 

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

"O " document refernng to an oral disclosure, use, exhibition or 
other means 

"P" document published prior to the international tiling date but 
later than the priority date claimed 



T" later document published atter the international filing date 
or priority date and not in conflict v^rith the application but 
cited to understand the principle or theory underlying the 
invention 

"X" document of particular relevance; the claimed invention 
cannot be considered novel or canrwt be considered to 
involve an inventive step when the document is taken alone 

'Y" documem of particular retevance; the claimed invention 

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

document member of the same patent family 



Date of the actual completion of theinterrtational search 



29 July 1998 



Date of mailing of the tmernationai search repon 



07/08/1998 



Name and mailing address of the ISA 

European Patent Office, P.B. 5818 Patentlaan2 
NL *a280 HV Rtjswijk 
Tel. (+31-70) 340-2040, Tx. 31 651 epo nl. 
Fax: (+31-70) 340-3016 



Authorized officer 



ESPINOSA, W 



Forni PCT/ISa«io (second sheet) (July 1992) 



page 1 of 2 



INTERNATIONAL SEARCH REPORT 



/national Application No 

PCT/US 98/04904 



C.<Cont(nuatlon) DOCUMENTS CONSIDERED TO BE RELEVANT 



Category 



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



Relevant to claim No. 



DATABASE WPI 

Section Ch, Week 8911 

Derwent Publications Ltd., London, GB; 

Class D22, AN 89-080728 

XP002073037 

& JP 01 032 371 A (NITTA GELATIN KK) 
see abstract 

WO 92 13565 A (SHAW ROBERT F) 20 August 
1992 

see claims; examples 

EP 0 147 021 A (GEISTLICH SOEHNE AG) 3 
July 1985 

see page 4, line 11 - line 12; claims 

DATABASE WPI 

Section Ch, Week 8835 

Derwent Publications Ltd., London, GB; 

Class 022, AN 88-247568 

XP002073038 

& JP 63 181 770 A (NAGASE M) 
see abstract 

DATABASE WPI 

Section Ch, Week 9001 

Derwent Publications Ltd., London, GB; 

Class A96, AN 90-004589 

XP002073039 

& JP 01 288 269 A (TOA NENRYO KOGYO KK) 
see abstract 

DATABASE WPI 

Section Ch, Week 9325 

Derwent Publications Ltd., London, GB; 

Class A96, AN 93-199779 

XP002073040 

& JP 05 123 390 A (KYOCERA CORP) 
see abstract 

DATABASE WPI 

Section Ch, Week 9347 

Derwent Publications Ltd., London, GB; 

Class A96, AN 93-373633 

XP002073041 

& JP 05 277 174 A (KYOCERA CORP) 
see abstract 



1,2 



1,2 



Form PCT/tSA/210 (continuation ot second sheet) (Jt4y 1982) 



page 2 of 2 



INTERNATIONAL SEARCH REPORT 



International application No. 

PCT/US 98/04904 



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

This International Search Report has not been established in respect of certain claims under Article I7<2)(a) for the following reasons: 



□ 



Claims Nos.: 29*32 

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

Remark: Although claims 29-32 

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. 

Claims Nos.: 

because they relate to parts of the international Application that do not comply with the prescribed requirements losuch 
an extent that no meaningful International Search can be earned out. specifically: 



I I Claims Nos.; 

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



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



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



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

^- D ^" searchable claims could be searched without effort justifying an additional tee. this Authority did not inviteoavment 
of any additional fee. 



^' rn ^^'"^ °* ^® required additional search tees were timely paid by the applicant, this Interrwtional Search Report 

— covers only those claims for which tees were paid. specifically claims Nos.: 



Q No required additional search fees were timely paid by the applicant. Conseauently. this Interr^tional Search Report is 
restricted to the invention first mentioned in the claims; it is covered by claims Nos.: 



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

j I No protest accompanied the payment of additional search lees. 



Form PCT/lSA/210 {continuation of fkst sheet {i))(July 1992) 



INTERNATIONAL SEARCH REPORT 

Information on patent family members 



rnational Ap|>tlcatlon No 

PCT/US 98/04904 



Patent document 




Publication 


Patent familv 




Publication 


cited in search report 




dale 




member(s) 




date 


WO 9639203 


A 


12-12-1996 


AU 




A 


24-12-1996 








FP 


0ftR177? 

\JOiJ 1 / / L 


A 


08-07-1998 


EP 0530804 


A 


10-03-1993 


U O 




A 


14-12-1993 








All 


oo / ooo 


B 


23-03-1995 








All 




A 


05-04-1993 








CA 


2116859 


A 


18-03-1903 








IL 


102988 


A 


08-02-1998 








IP 

jr 


7cnn7/i 1 


T 


26-01-1995 








NO 


940764 


A 


20-04-1004 








NZ 


244060 


A 


27-07-1997 








WO 


9304710 


A 


18-03-1993 








ZA 


9206729 


A 


12-03-1993 


EP 0329239 


A 


23-08-1989 


.IP 


tvUDrDU 


A 










IK 




A 




OE 4216496 


A 


25-11-1993 


riuiMt 








WO 9213565 


A 


20-08-1992 


lie 




A 










AT 




T 

1 










Al 1 




R 
u 










All 
MU 


1/11 OQQO 


A 


U/ l33C 








PA 


5 1 m cc£ 
^: lUibbo 


A 

n 


m -nft-1 009 












A 


'^n-HQ-l 009 














uo uo 1373 














n4-m -1 ooA 

UH UJ. 11770 








ni^ 

UN 




T 


1 n-n7-i ooR 

lU U/ A3?3 








EP 


0569541 


A 


1Q-1 "I-IQO'^ 
lO X 1 X 770 








ES 


2072144 


T 


01-07-1995 








i L 


0/Dib 


B 


03-04-1996 








7 1 
i L 


1 nn70Q 
luu/yy 


A 


1 n-1 00^ 

lO XU X370 








IP 

or 


ObUD^Do 




lo uo iyy*f 












A 


C / U/ X J7 / 








US 


5368858 


A 


29-11-1994 


EP 0147021 


A 


03-07-1985 


AU 


3461484 


A 


09-05-1985 








CA 


1250237 


A 


21-02-1989 








JP 


60103963 


A 


^08-06-1985 








US 


4772468 


A 


20-09-1988 



Form PCT/ISA^iO (patem family annex) (July 1992) 



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