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Europaisches Patentamt 
Qjjt European Patent Office 

Office europeen des brevets 




© Publication number: 



0 530 804 A1 



EUROPEAN PATENT APPLICATION 



© Application number: 92115079.3 
(§) Date of filing: 03.09.92 



© Int. CIA A61L 25/00, A61 L 27/00, 
A61K 37/02 



© Priority: 06.09.91 US 756164 


© Applicant: Shaw, Robert Francis 


@ Date of publication of application: 


1750 Taylor Avenue, Penthouse 2401 


San Francisco, CA 94133(US) 


10.03.93 Bulletin 93/10 




© Designated Contracting States: 


© Inventor: Hunzlker, Ernst B. 


Sonnenrain 32 


AT BE CH DE DK ES FR GB GR IE IT LI LU MC 


CH-4533 Rledholz(CH) 


NL PT SE 






© Representative: Vossius & Partner 




Siebertstrasse 4 P.O. Box 86 07 67 




W-8000 Munchen 86 (DE) 



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© Kits and compositions for the treatment and repair of defects or lesions In cartilage or bone. 



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© Kits and compositions are provided for the treat- 
ment and repair of defects in the cartilage or bone of 
humans and other animals as in full-thickness de- 
fects in joints. A matrix having pores large enough to 
allow cells to populate the matrix and to form blood 
vessels is used to fill the defect in bone. The matrix 
filling the bone defect contains an angiogenic factor 
and also contains an osteogenic factor in an appro- 
priate delivery system. A matrix having pores suffi- 
ciently large to allow cartilage repair cells to popu- 
late the matrix is used to fill a defect in cartilage to 
induce cartilage formation. The matrix filling the de- 
fect in cartilage contains a proliferation agent and 
also contains a transforming factor in an appropriate 
delivery system. The matrix may also contain a 
chemotactic agent to attract cartilage repair cells. In 
a full-thickness defect, a membrane is used to sepa- 
rate the defect sites in bone and cartilage, which is 
sealed to the cartilage-bone-junction and which pre- 
vents blood vessels and associated cells from pene- 
trating from one site to the other. 



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Rank Xerox (UK) Business Services 

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This invention relates to pharmaceutical com- 
positions for the treatment and repair of defects or 
lesions in cartilage or bone. More specifically, this 
invention relates to pharmaceutical compositions 
for treating defects or lesions (used interchange- 
ably herein) in cartilage or bone and to composi- 
tions comprising a matrix containing one or more 
proliferating agents and a transforming factor to 
promote proliferation and transformation of carti- 
lage repair cells to form new stable cartilage tissue 
and to compositions comprising a matrix containing 
an angiogenic factor to stimulate blood vessel for- 
mation and an osteogenic factor to stimulate forma- 
tion of bone. The compositions of this invention are 
particularly useful in the treatment of full-thickness 
defects found in severe osteoarthritis, and in other 
diseases and traumas that produce cartilage or 
bone injury. 

Joints are one of the common ways bones in 
the skeleton are connected. The ends of normal 
articulated bones are covered by articular cartilage 
tissue, which permits practically frictionless move- 
ment of the bones with respect to one another [L. 
Weiss, ed., Cell and Tissue Biology (Munchen: 
Urban and Schwarzenburg, 1988) p. 247]. 

Articular cartilage is characterized by a particu- 
lar structural organization. It consists of specialized 
cells (chondrocytes) embedded in an intercellular 
material (often . referred to in the literature as the 
"cartilage matrix") which is rich in proteoglycans, 
collagen fibrils of predominantly type II, other pro- 
teins, and water [Buckwalter et al., "Articular Car- 
tilage: Injury and Repair," in Injury and Repair of 
the Musculoskeletal Soft Tissues (Park Ridge, III.: 
American Academy of Orthopaedic Surgeons Sym- 
posium, 1987) p. 465]. Cartilage tissue is neither 
innervated nor penetrated by the vascular or lym- 
phatic systems. However, in the mature joint of 
adults, the underlying subchondral bone tissue, 
which forms a narrow, continuous plate between 
the bone tissue and the cartilage, is innervated and 
vascularized. Beneath this bone plate, the bone 
tissue forms trabeculae, containing the marrow. In 
immature joints, articular cartilage is underlined by 
only primary bone trabeculae. A portion of the 
meniscal tissue in joints also consists of cartilage 
whose make-up is similar to articular cartilage 
[Beaupre, A. et al., Clin. Orthop. Rel. Res. , pp. 72- 
76 (1 986)]. 

Two types of defects are recognized in articu- 
lar surfaces, i.e., full-thickness defects and superfi- 
cial defects. These defects differ not only in the 
extent of physical damage to the cartilage, but also 
in the nature of the repair response each type of 
lesion can elicit. . 

Full-thickness defects of an articular surface 
include damage to the hyaline cartilage, the cal- 
cified cartilage layer and the subchondral bone 



tissue with its blood vessels and bone marrow. 
Full-thickness defects can cause severe pain since 
the bone plate contains sensory nerve endings. 
Such defects generally arise from severe trauma or 

5 during the late stages of degenerative joint disease, 
such as osteoarthritis. Full-thickness defects may, 
on occasion, lead to bleeding and the induction of 
a repair reaction from the subchondral bone 
[Buckwalter et al., "Articular Cartilage: Composition, 

70 Structure, Response to Injury, and Methods of Fa- 
cilitating Repair," in Articular Cartilage and Knee 
Joint Function: Basic~Science and Arthroscopy - 
(New York: Raven Press, 1990) pp. 19-56]. The 
repair tissue formed is vascularized fibrous type of 

75 cartilage with insufficient biomechanical properties, 
and does not persist on a long-term basis 
[Buckwalter et al. (1990). supra ]. 

Superficial defects in the articular cartilage tis- 
sue are restricted to the cartilage tissue itself. Such 

20 defects are notorious because they do not heal and 
show no propensity for repair reactions. 

Superficial defects may appear as fissures, 
divots, or clefts in the surface of the cartilage, or 
they may have a "crab-meat" appearance in the 

25 affected tissue. They contain no bleeding vessels 
(blood spots) such as are seen in full-thickness 
defects. Superficial defects may have no known 
cause, but often they are the result of mechanical 
derangements which lead to a wearing down of the 

30 cartilaginous tissue. Mechanical derangements may 
be caused by trauma to the joint, e.g., a displace- 
ment of torn meniscus tissue into the joint, 
meniscectomy, a taxation of the joint by a torn 
ligament, malalignment of joints, or bone fracture, 

35 or by hereditary diseases. Superficial defects are 
also characteristic of early stages of degenerative 
joint diseases, such as osteoarthritis. Since the 
cartilage tissue is not innervated [Ham's Histology - 
(9th ed.) (Philadelphia: J.B. Lippincott Co. 1987), 

40 pp. 266-272] or vascularized, superficial defects 
are not painful. However, although painless, su- 
perficial defects do not heal and often degenerate 
into full-thickness defects. 

It is generally believed that because articular 

45 cartilage lacks a vasculature, damaged cartilage 
tissue does not receive sufficient or proper stimuli 
to elicit a repair response [Webber et al., "Intrinsic 
Repair Capabilities of Rabbit Meniscal Fibrocar- 
tilage: A Cell Culture Model", {30th Ann. Orthop. 

so Res. Soc, Atlanta, Feb. 1984); Webber et al., J. 
Orthon. Res. , 3, pp. 36-42 (1985)]. It is theorized 
that the chondrocytes in the cartilaginous tissue are 
normally not exposed to sufficient amounts of 
repair-stimulating agents such as growth factors 

55 and fibrin clots typically present in damaged 
vascularized tissue. 

One approach that has been used to expose 
damaged cartilage tissue to repair stimuli involves 



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drilling or scraping through the cartilage into the 
subchondral bone to cause bleeding [Buckwalter et 
al. (1990), supra]. Unfortunately, the repair re- 
sponse of the tissue to such surgical trauma is 
usually comparable to that observed to take place 
naturally in full-thickness defects that cause bleed- 
ing, viz., formation of a fibrous type of cartilage 
which exhibits insufficient biomechanical properties 
and which does not persist on a long-term basis 
[Buckwalter et al. (1990), supra], 

A variety of growth factors have been isolated 
and are now available for research and biomedical 
applications [see e.g., Rizzino, A., Dev. Biol., 130, 
pp. 411-422 (1988)]. Some of these growth factors, 
such as transforming growth factor beta (TGF-j8), 
have been reported to promote formation of 
cartilage-specific molecules, such as type II col- 
lagen and cartilage-specific proteoglycans, in em- 
bryonic rat mesenchymal cells in vitro [e.g., 
Seyedin et al., Proc. Natl. Acad. Sci. USA , 82, pp. 
2267-71 (1985); Seyedin et al., J. Biol. ChemT261 , 
pp. 5693-95 (1986); Seyedin et al., J. Biol. Chem ., 
262 , pp. 1946-1949 (1987)]. 

Furthermore, a number of protein factors have, 
been identified that apparently stimulate formation 
of bone. Such osteogenic factors include bone 
morphogenetic proteins, osteogenin, bone 
osteogenic protein (BOP), TGF-j8s, and recombin- 
ant bone inducing proteins. 

Millions of patients have been diagnosed as 
having osteoarthritis, i.e., as having degenerating 
defects or lesions in their articular cartilage. Never- 
theless, despite claims of various methods to elicit 
a repair response in damaged cartilage, none of 
these treatments has received substantial applica- 
tion [Buckwalter et al. (1990), supra; Knutson et al., 
J. Bone and Joint Surg. , 68-B, p. 795 (1986); 
Knutson et al., J. Bone and Joint Surg., 67-B, p. 47 
(1985); Knutson et al., Clin. Orthop. , 19l7 ~p. 202 
(1984); Marquet, Clin. Orthop. , 146 , p. T02 (1980)]. 
And such treatments have generally provided only 
temporary relief. Systemic use of 
"chondroprotective agents" has also been purport- 
ed to arrest the progression of osteoarthritis and to 
induce relief of pain. However, such agents have 
not been shown to promote repair of lesions or 
defects in cartilage tissue. 

To date, treatment of patients suffering from 
osteoarthritis has been directed largely to symp- 
tomatic relief through the use of analgesics and 
anti-inflammatory agents. Without a treatment that 
will elicit repair of superficial defects in articular 
cartilage, the cartilage frequently wears down to the 
subchondral bone plate. At this phase of the dis- 
ease, i.e., severe osteoarthritis, the unremitting na- 
ture of the pain and the significant compromise of 
function often dictates that the entire joint be ex- 
cised and replaced with an artificial joint of metal 



and/or plastic. Some one-half million procedures 
comprising joint resection and replacement with an 
artificial joint are currently performed on knees and 
hips each year. [See e.g., Graves, E. J., "1988 

5 Summary; National Hospital Discharge Survey", 
Advanced Data From Vital and Health Statistics , 
185, pp. 1-12 (June 19, 1990)]. 

There is, therefore, a need for a reliable treat- 
ment for cartilage in superficial cartilage defects, 

w e.g., as found in the early stages of osteoarthritis. 
There is also a needs for treatment of cartilage or 
bone defects as found in the lesions of severe 
osteoarthritis and for the treatment of other bone 
defects. 

75 The present invention solves the problems re- 
ferred to above by providing effective therapeutic 
kits and compositions to induce the repair of le- 
sions in cartilage or bone of humans and other 
animals. Use of the kits and compositions of this 

20 invention also promote the healing of traumatic 
lesions and forms of osteoarthritis which would 
otherwise lead to loss of effective joint function 
leading to probable resection and replacement of 
the joint. 

25 In general outline, the kits of this invention for 

repairing full-thickness defects in joints a matrix 
that will be incorporated into the animal tissue and 
is generally biodegradable which may be used for 
filling the defect in the bone portion of a full- 

30 thickness defect up to the level of the bone-car- 
tilage interface. The kit also comprises a mem- 
brane, which is impermeable to ceils and may be 
used to cover the matrix filling the bone defect. 
The membrane is sealed to the edges of the defect 

35 at the cartilage-bone junction, e.g., by sealing to 
the cartilage by thermal bonding using a thermal 
knife or laser. The kit further comprises a matrix 
which contains a chondrogenic composition, and 
which will be incorporated into the animal tissue 

40 and is generally biodegradable and may be used to 
fill the remaining, cartilage portion of the defect to 
the top of the cartilage surface. The matrix contain- 
ing angiogenic and osteogenic factors may also be 
applied to any bone defect to promote repair. The 

45 compositions of this invention for repairing bone 
defects that do not involve cartilage, are such that 
the bone defect is filled with a composition com- 
prising a matrix containing angiogenic factor(s) and 
osteogenic factor(s). The osteogenic factor(s) is 

50 packaged in an appropriate delivery system. 

The kits and compositions for the treatment of 
full-thickness defects can be used during arth- 
roscopic, open surgical or percutaneous proce- 
dures. Certain kits of this invention may be used 

55 such that after identification of the defect, (1) a 
composition comprising a matrix containing an an- 
giogenic factor and an osteogenic factor packaged 
in an appropriate delivery system, e.g., liposomes, 



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is used for filling the bone portion of the defect; (2) 
a membrane, preferably a biodegradable mem- 
brane, which prevents cells from migrating from the 
bone defect side to the cartilage defect side, is 
used by placing it over the matrix in the bone 
defect and sealing the membrane to the edges of 
the defect at the cartilage-bone junction; and (3) a 
composition comprising a matrix, preferably 
biodegradable, and containing a proliferation agent 
and a transforming factor which is packaged in an 
appropriate delivery system is used for filling the 
cartilage portion of the defect. 

In this last step, the matrix is bonded to the 
surface of the cartilage portion of the full-thickness 
defect, for example, by using an adhesion-promot- 
ing factor, such as transglutaminase. 

In order that the invention may be more fully 
understood, the following detailed description is 
provided. In the description the following terms are 
used. - 

Angiogenic Factor -- as used herein, refers to 
any peptide, polypeptide, protein or any other com- 
pound or composition which induces or stimulates 
the formation of blood vessels and associated cells 
(such as endothelial, perivascular, mesenchymal 
and smooth muscle cells) and blood vessel-asso- 
ciated basement membranes. In vivo and in vitro 
assays for angiogenic factors are well-known in the 
art [e.g., Gimbrone, M. A., et al., J. Natl. Cancer 
Inst. , 52, pp. 413-419 (1974); Klagsbrun, M. et al., 
Cancer Res. , 36, pp. 110-113 (1976); Gross et al., 
Proc. Natl. Acad. Sci. (USA) , 80, pp. 2623-2627 
(1983); Gospodarowicz et al., Proc. Natl. Acad. Sci. 
(USA) , 73, pp. 4120-4124 (1976); Folkman et al., 
Proc. Natl. Acad. Sci. (USA) , 76, pp. 5217-5221 
(1979); Zetter, B. R., Nature (London) , 285 , pp. 41- 
43 (1980); Azizkhan, R. G. et al., J. Exp. Med. , 152 , 
pp. 931-944 (1980)]. 

Arthroscopy - as used herein, refers to the use 
of an arthroscope to examine or perform surgery 
on a joint. 

Bone as used herein, refers to a calcified 
connective tissue primarily comprising a network of 
deposited calcium and phosphate in the form of 
hydroxyapatite, collagen (predominantly type I col- 
lagen) and bone cells, such as osteoblasts and 
osteoclasts. 

Bone Repair Cell -- as used herein, refers to a 
cell which, when exposed to appropriate stimuli, 
will differentiate and be transformed into a bone 
cell, such as an osteoblast or an osteocyte, which 
forms bone. Bone repair cells include perivascular 
cells, mesenchymal cells, fibroblasts, fibroblast-like 
cells and dedifferentiated chondrocytes. 

Cartilage -- as used herein, refers to a type of 
connective tissue that contains chondrocytes em- 
bedded in an intercellular material (often referred to 
as the "cartilage matrix") comprising fibrils of col- 



lagen (predominantly type II collagen along with 
other minor types, e.g., types IX and XI), various 
proteoglycans (e.g., chondroitinsulfate-, 
keratansulfate-, and dermatansulfate prot- 

5 eoglycans), other proteins, and water. Cartilage as . 
used herein includes articular and meniscal car- 
tilage. Articular cartilage covers the surfaces of the 
portions of bones in joints and allows movement in 
joints without direct bone-to-bone contact, and 

70 thereby prevents wearing down and damage to 
apposing bone surfaces. Most normal healthy ar- 
ticular cartilage is also described as "hyaline", i.e., 
having a characteristic frosted glass appearance. 
Meniscal cartilage is usually found in joints which 

75 are exposed to concussion as well as movement. 
Such locations of meniscal cartilage include the 
temporo-mandibular, sterno-clavicular, acromio- 
clavicular, wrist and knee joints [Gray's Anatomy - 
(New York: Bounty Books, 1977)]. 

20 Cartilage Repair Cell -- as used herein, refers 

to a cell which, when exposed to appropriate stim- 
uli, will differentiate and be transformed into a 
chondrocyte. Cartilage repair cells include mesen- 
chymal cells, fibroblasts, fibroblast-like cells, 

25 macrophages and dedifferentiated chondrocytes. 

Cell Adhesion Promoting Factor - as used 
herein, refers to any compound or composition, 
including fibronectin and other peptides as small as 
tetrapeptides which comprise the tripeptide Arg- 

30 Gly-Asp, which mediates the adhesion of cells to 
extracellular material [Ruoslathi et a!., Cell, 44, pp. 
517-518(1986)]. 

Chemotactic Agent — as used herein, refers to 
any compound or composition, including peptides, 

35 proteins, glycoproteins and glycosaminoglycan 
chains, which is capable of attracting cells in stan- 
dard in vitro chemotactic assays [e.g., Wahl et al., 
Proc. Natl. Acad. Sci. USA , 84. pp 5788-92 (1987); 
Postlewaite et al., J. Exp. Med. , 165 , pp. 251-56 

AO (1987); Moore et al., Int. J. Tiss. Reaa, XI, pp. 301- 
07(1989)]. 

Chondrocytes - as used herein, refers to cells 
which are capable of producing components of 
cartilage tissue, e.g., type II cartilaginous fibrils and 

45 fibers and proteoglycans. 

Fibroblast growth factor (FGF) - any member 
of the family of FGF polypeptides [Gimenez-Gal- 
lego et al., Biochem. Biophys. Res. Commun., 135, 
pp. 541-548 (1986); Thomas it aT Trends 

50 Biochem. Sci. , I^, pp. 81-84 (1986)] or derivatives 
thereof, obtained from natural, synthetic or recom- 
binant sources, which exhibits the ability to stimu- 
late DNA synthesis and cell division in vitro [for 
assays see, e.g., Gimenez-Gallego et al., 1986, 

55 supra ; Canalis et al., J. Clin. Invest. , 81, pp. 1572- 
1577 (1988)] of a variety of cells, including primary 
fibroblasts, chondrocytes, vascular and corneal en- 
dothelial cells, osteoblasts, myoblasts, smooth 



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muscle and glial cells [Thomas et al., 1986, supra]. 
FGFs may be classified as acidic (aFGF) or basic 
(bFGF) FGF, depending on their isoelectric points 
(Pi). 

Matrix as used herein, refers to a porous 
composite, solid or semi-solid substance having 
pores or spaces sufficiently large to allow cells to 
populate the matrix. The term matrix includes 
matrix-forming materials, i.e., materials which can 
form matrices within a defect site in cartilage or 
bone. Matrix-forming materials may require addi- 
tion of a polymerizing agent to form a matrix, such 
as adding thrombin to a solution containing 
fibrinogen to form_a fibrin matrix. Other matrix 
materials include collagen, combinations of col- 
lagen and fibrin, agarose (e.g., Sepharose®), and 
gelatin. Calcium phosphate may be used alone or 
in combination with other matrix materials in treat- 
ing defects in bones. 

Membrane - as used herein, refers to any 
material which can be placed between the bone 
defect portion and the cartilage defect portion of a 
full thickness defect and which prevents cell migra- 
tion and blood vessel infiltration from the bone 
defect portion into the cartilage defect portion of 
the full thickness defect. The membranes used in 
the methods and compositions of this invention for 
the repair of full thickness defects are preferably 
biodegradable. 

Osteogenic Factor -- as used herein, refers to 
any peptide, polypeptide, protein or any other com- 
pound or composition which induces or stimulates 
the formation of bone. The osteogenic factor in- 
duces differentiation of bone repair cells into bone 
cells, such as osteoblasts or osteocytes. This pro- 
cess may be reached via an intermediary state of 
cartilage tissue. The bone tissue formed from bone 
cells will contain bone specific substances such as 
type I collagen fibrils, hydroxyapatite mineral and 
various glycoproteins and small amounts of bone 
proteoglycans. 

Proliferation (mitogenic) Agent -- as used here- 
in, refers to any compound or composition, includ- 
ing peptides, proteins, and glycoproteins, which is 
capable of stimulating proliferation of cells in vitro. 
In vitro assays to determine the proliferation 
(mitogenic) activity of peptides, polypeptides and 
other compounds are well-known in the art [see, 
e.g., Canalis et al., J. Clin. Invest., pp. 1572-77 
(1988); Gimenez-Gallego et al., Biochem. Biophys. 
Res. Commun., 135, pp. 541-548 (1986); Rizzino, 
"Soft Agar GrowthAssays for Transforming Growth 
Factors and Mitogenic Peptides", in Methods En- 
zymol., 146A (New York: Academic Press, 1987), 
pp. 341-52; Dickson et al., "Assay of Mitogen- 
Induced Effects on Cellular Incorporation of Precur- 
sors for Scavengers, de Novo, and Net DNA Syn- 
thesis", in Methods "Enzymol., 146A (New York: 



Academic Press, 1987), pp. 329-40]. One standard 
method to determine the proliferation (mitogenic) 
activity of a compound or composition is to assay it 
in vitro for its ability to induce anchorage-indepen- 

5 dent growth of nontransformed cells in soft agar 
[e.g., Rizzino, 1987, supra]. Other mitogenic activity 
assay systems are also known [e.g., Gimenez- 
Gallego et al., 1986, supra; Canalis et al., 1988, 
supra; Dickson et al., 1987, supra]. Mitogenic ef- 

io fects of agents are frequently very concentration- 
dependent, and their effects can be reversed at 
lower or higher concentrations than the optimal 
concentration range for mitogenic effectiveness. 
Transforming Factor -- as used herein, refers to 

75 any peptide, polypeptide, protein, or any other 
compound or composition which induces differenti- 
ation of a cartilage repair cell into a chondrocyte. 
The ability of the compound or composition to 
induce or stimulate production of cartilage-specific 

20 proteoglycans and type II collagen by cells can be 
determined by in vitro assays known in the art 
[Seyedin et al., Proc. Natl. Acad. Sci. USA , 82, pp. 
2267-71 (1985); Seyedin et al., Path. Immunol. 
Res. , 7, pp. 38-42 (1987)]. 

25 Transforming Growth Factor Beta (TGF-0) - 

-any member of the family of TGF-/3 polypeptides 
[Derynck, R. et al., Nature, 316, pp. 701-705 
(1985); Roberts et al., "The transforming growth 
factory's", In Peptide growth factors and their re- 

30 ceptors I (Berlin: Springer Verlag, 1990), p. 419)] or 
derivatives thereof, obtained from natural, synthetic 
or recombinant sources, which exhibits the char- 
acteristic TGF-jS ability to stimulate normal rat kid- 
ney (NRK) cells to grow and form colonies in a soft 

35 agar assay [Roberts et al., "Purification of Type £ 
Transforming Growth Factors From Nonneoplastic 
Tissues", in Methods for Preparation of Media, 
Supplements, and Substrata for Serum-Free Animal 
Cell Culture (New York: Alan R. Liss, Inc., 1984)] 

40 and which is capable of inducing transformation of 
cartilage repair cells into chondrocytes as eviden- 
ced by the ability to induce or stimulate production 
of cartilage-specific proteoglycans and type II col- 
lagen by cells in vitro [Seyedin et al., 1985, supra]. 

45 This invention relates to compositions and kits 

for treating defects or lesions in cartilage or bone. 
The compositions of this invention comprise ma- 
trices having pores sufficiently large to allow cells 
to populate the matrices. 

so For use in the repair of cartilage as in superfi- 
cial defects or the cartilage layer in a full-thickness 
defect, the matrix will also contain a proliferation 
agent to stimulate the proliferation of cartilage re- 
pair cells in the matrix. Preferably, the proliferation 

55 agent also serves as a chemotactic agent to attract 
cartilage repair cells to the matrix. Alternatively, the 
matrix may contain a chemotactic agent in addition 
to the proliferation agent. In one preferred embodi- 



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ment of this invention, the matrix also contains an 
appropriate concentration of a transforming factor, 
the transforming factor being contained within or in 
association with a delivery system which effects 
release of the transforming factor at the appropriate 
time to transform the proliferated cartilage repair 
cells in the matrix into chondrocytes which pro- 
duce, stable cartilage tissue. The matrix may also 
contain a cell adhesion promoting factor. 

Matrix materials useful in the methods and 
compositions of this invention for filling or other- 
wise dressing the cartilage or bone defects include 
fibrinogen (activated with thrombin to form fibrin in 
the defect or lesion), collagen, agarose, gelatin and 
any other biodegradable material which forms a 
matrix with pores sufficiently large to allow cartilage 
or bone repair cells to populate and proliferate 
within the matrix and which can be degraded and 
replaced with cartilage or bone during the repair 
process. In some instances, calcium phosphate 
containing compounds may be used alone or in 
combination with other biodegradable matrix ma- 
terials in treating bone defects. 

The matrices useful in the compositions and 
kits of this invention may be preformed or may be 
formed in situ, for example, by polymerizing com- 
pounds and compositions such as fibrinogen to 
form a fibrin matrix. Matrices that may be prefor- 
med include collagen (e.g., collagen sponges and 
collagen fleece), chemically modified collagen, 
gelatin beads or sponges, a gel-forming substance 
such as agarose, and any other gel-forming or 
composite substance that is composed of a matrix 
material that will fill the defect and allow cartilage 
or bone repair cells to populate the matrix, or 
mixtures of the above. 

In one embodiment of this invention, the matrix 
is formed using a solution of fibrinogen, to which is 
added thrombin to initiate polymerization shortly 
before use. A fibrinogen concentration of 0.5-5 
mg/ml of an aqueous buffer solution may be used. 
Preferably, a fibrinogen solution of 1 mg/ml of an 
aqueous buffer solution is used. Polymerization of 
this fibrinogen solution in the defect area yields a 
matrix with a pore size sufficiently large (e.g., ap- 
proximately 50-200 urn) so that cartilage or bone 
repair cells are free to populate the matrix and 
proliferate in order to fill the volume of the defect 
that the matrix occupies. Preferably, a sufficient 
amount of thrombin is added to the fibrinogen 
solution shortly before application in order to allow 
enough time for the surgeon to deposit the material 
in the defect area prior to completion of poly- 
merization. Typically, the thrombin concentration 
should be such that polymerization is achieved 
within a few to several (2-4) minutes since expo- 
sure of cartilage to air for lengthy periods of time 
has been shown to cause damage [Mitchell et al., 



J. Bone Joint Surg. , 71 A , pp. 89-95 (1989)]. Exces- 
sive amounts of thrombin should not be used since 
thrombin has the ability to cleave growth factor 
molecules and inactivate them. Thrombin solutions 

5 of 10-500 units per ml, and preferably 100 units 
per ml, of an aqueous buffer solution may be 
prepared for addition to the fibrinogen solution. In a 
preferred embodiment of this invention, approxi- 
mately 20 ul of thrombin (100 U/ml) are mixed with 

io each ml of a fibrinogen solution (1 mg/ml) approxi- 
mately 200 seconds before filling the defect. Poly- 
merization will occur more slowly if a lower con- 
centration of thrombin is added. It will be appre- 
ciated that the amount of thrombin solution needed 

75 to achieve fibrin polymerization within 2-4 minutes 
can be given only approximately, since it depends 
upon the environmental temperature, the tempera- 
ture of the thrombin solution, the temperature of 
the fibrinogen solution, etc. The polymerization of 

20 the throm bin-activated matrix solution filling the de- 
fect is easily monitored by observing the thrombin- 
induced polymerization of an external sample of 
the fibrinogen solution. Preferably, in the composi- 
tions and methods of this invention, fibrin matrices 

25 are formed from autologous fibrinogen molecules, 
i.e., fibrinogen molecules derived from the blood of 
the same mammalian species as the species to be 
treated. Non-immunogenic fibrinogen from other 
species may also be used. 

30 Matrices comprising fibrin and collagen may 

also be used in the compositions and kits of this 
invention. In a preferred embodiment of this inven- 
tion, collagenous matrices are used. 

When collagen is used as a matrix material, 

as sufficiently viscous solutions can be made, e.g., 
using Collagen-Vliess® ("fleece"), Spongostan®, or 
gelatine-blood-mixtures, and there is no need for a 
polymerizing agent. Collagen matrices may also be 
used with a fibrinogen solution activated with a 

40 polymerizing agent so that a combined matrix re- 
sults. 

Polymerizing agents may also be unnecessary 
when other biodegradable compounds are used to 
form the matrix. For example, Sepharose® solu- 

45 tions may be chosen that will be liquid matrix 
solutions at 39-42 *C and become solid (i.e., gel- 
like) at 35-38 • C. The Sepharose should also be at 
concentrations such that the gel filling the defect 
has a mesh size to allow bone or cartilage repair 

so ceils to freely populate the matrix and defect area. 

In the compositions of this invention used in 
cartilage repair, one or more proliferation 
(mitogenic) agents may be added to the matrix 
solution. The proliferation agent or agents should 

55 be present in an appropriate concentration range to 
have a proliferative effect on cartilage repair cells 
in the matrix filling the defect. Preferably, the same 
agent should also have a chemotactic effect on the 



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cells (as in the case of TGF-0); however, a factor 
having exclusively a proliferative effect may be 
used. Alternatively, to produce chemotactic cell im- 
migration, followed by induction of cell proliferation, 
two different agents may be used, each one having 
just one of those specific effects (either chemotac- 
tic or proliferative). 

Proliferation (mitogenic) agents useful in the 
compositions and kits of this invention for stimulat- 
ing the proliferation of cartilage repair cells include 
transforming growth factors ("TGFs") such as TGF- 
as and TGF-jSs; insulin-like growth factor ( M IGF I"); 
acidic or basic fibroblast growth factors ("FGFs"); 
platelet-derived growth factor ("PDGF"); epidermal 
growth factor ("EGF"); and hemopoietic growth fac- 
tors, such as interleukin 3 ("IL-3") [Rizzino, 1987, 
supra; Canalis et al., supra, 1988; Growth factors in 
biology and medicine, Ciba Foundation Sympo- 
sium, 116 (New York: John Wiley & Sons, 1985); 
BasergI7~R., ed., Cell growth and division (Oxford: 
IRL Press, 1985); Sporn, MA and Roberts, A.B., 
eds., Peptide growth factors and their receptors, 
Vols. I and II (Berlin: Springer-Verlag, 1990)]. How- 
ever, these particular examples are not limiting. 
Any compound or composition which is capable of 
stimulating the proliferation of cells as demonstrat- 
ed by an in vitro assay for cell proliferation is 
useful as a proliferation agent in this invention. 
Such assays are known in the art [e.g., Canalis et 
al., 1988, supra ; Gimenez-Gallego et al., 1986, 
supra ; Dickson et al., 1987, supra ; Rizzino, 1987, 
supra ]. 

Chemotactic agents useful in the compositions 
and kits of this invention for attracting cartilage 
repair cells to the cartilage defect incjude, for ex- 
ample, TGF-jSs, FGFs (acid or basic), PDGF, tumor 
necrosis factors (e.g., TNF-a, TNF-/8) and prot- 
eoglycan degradation products, such as 
glycosaminoglycan chains [Roberts et al. (1990), 
supra; Growth factors in biology and medicine, 
Ciba Foundation Symposium, 116 (New York, John 
Wiley & Sons, 1985); R. BaseTga, ed., Cell growth 
and division (Oxford: IRL Press, 1985)]. Assays to 
determine the chemotactic ability of polypeptides 
and other compounds are known in the art [e.g., 
Postlewaite et al., 1987, supra ; Wahl et al., 1987, 
supra; Moore et al., 1989, supra]. 

In a preferred embodiment of this invention, the 
matrix used in cartilage repair contains TGF-0 as 
the proliferation agent and as the chemotactic 
agent. In particular, TGF-0I or TGF-0II may be 
used as the proliferation and chemotactic agent. 
Other TGF-iS forms (e.g.. TGF-0III, TGF-0IV, TGF- 
0V, etc.) or polypeptides having TGF-0 activity 
[see Roberts, 1990, supra ] may also be useful for 
this purpose, as well as other forms of this sub- 
stance to be detected in the future, and other 
growth factors. For use as the proliferation agent 



and chemotactic agent, TGF-0 molecules are dis- 
solved or suspended in the matrix at a concentra- 
tion of preferably 2-50 ng/ml of matrix solution, and 
most preferably, 2-10 ng/ml of matrix solution. It 
5 will be appreciated that the preferred concentration 
of TGF-0 that will stimulate proliferation of cartilage 
repair cells may vary with the particular animal to 
be treated. 

A transforming factor or factors may also be 
70 present in the matrix solution used in cartilage 
repair so that after cartilage repair cells have popu- 
lated the matrix, the transforming factor will be 
released into the defect site in a concentration 
sufficient to promote differentiation (i.e., transforma- 
75 tion) of the cartilage repair cells into chondrocytes 
which form new stable cartilage tissue. Proper tim- . 
ing of the release of the transforming factor is 
particularly important if the transforming factor can 
inhibit or interfere with the effectiveness of the 
20 proliferation agent [see Roberts et al. (1990), 
supra]. 

Transforming factors useful in the compositions 
and kits of this invention to promote cartilage repair 
include any peptide, polypeptide, protein or any 

25 other compound or composition which induces dif- 
ferentiation of cartilage repair cells into chon- 
drocytes which produce cartilage-specific prot- 
eoglycans and type II collagen. The ability of a 
compound or composition to induce or stimulate 

30 production of cartilage-specific proteoglycans and 
type II collagen in cells can be. determined using 
assays known in the art [e.g., Seyedin et al.. 1985, 
supra ; Seyedin et al., 1987, supra ]. The transform- 
ing factors useful in the compositions and kits of 

35 this invention include, for example, TGF-0S, TGF- 
as and FGFs (acid or basic). These transforming 
factors may be used singly or in combination. In 
addition, TGF-0 may be used in combination with 
EGF. 

40 The properly timed release of the transforming 

factor may be achieved by packaging the trans- 
forming factor in or with and appropriate delivery 
system. Delivery systems useful in the composi- 
tions and kits of this invention include liposomes, 

45 bioerodible polymers, carbohydrate-based corpus- 
cles, water-oil emulsions, fibers such as collagen 
which are chemically linked to heparin sulfate prot- 
eoglycans or other such molecules to which trans- 
forming factors bind spontaneously r and osmotic 

so pumps. Delivery systems such as liposomes, 
bioerodible polymers, fibers with bound transform- 
ing factors and carbohydrate-based corpuscles 
containing the transforming agent may be mixed 
with the matrix solution used to fill the defect. 

55 These systems are known and available in the art 
[see P. Johnson and J. G. Lloyd-Jones, eds., Drug 
Delivery Systems (Chichester, England: Ellis Hor- 
wood Ltd., 1987)]. Liposomes may be prepared 



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according to the procedure of Kim et al., Biochem. 
Biophys. Acta , 728, pp. 339-348 (1983). Other lipo- 
some preparation" procedures may also be used. 
Additional factors' for stimulating chondrocytes to 
synthesize the cartilage tissue components may be 
included with the transforming factor in the delivery 
system. 

In a preferred embodiment of this invention, the 
matrix used in cartilage repair contains TGF-0 as 
the proliferation and chemotactic agent, and con- 
tains TGF-/S packaged in a delivery system as the 
transforming factor. In particular, TGF-jSI or TGF-/JII 
may be used as the proliferation and chemotactic 
agent and as the transforming factor. Other TGF-0 
forms (e.g., TGF-/SIII, TGF-0IV, TGF-/SV, etc.) or 
polypeptides having TGF-0 activity (see Roberts, 
1990, supra ) may also be useful for this purpose, 
as well as other forms of this substance to be 
detected in the future, and other growth factors. 

In a preferred embodiment for cartilage repair, 
a TGF-/5 concentration of preferably 2-50 ng/ml of 
matrix solution, and most preferably, 2-10 ng/ml of 
matrix solution, is used as a proliferation agent and 
as a chemotactic agent. A substantially higher con- 
centration of TGF-/9 is also present in a subse- 
quently releasable form in the matrix composition 
as a transforming factor. Preferably, the subse- 
quent concentration of TGF-0 is greater than 200 
ng/ml of matrix and, most preferably, is greater 
than 500 ng/ml of matrix. It will be appreciated that 
the preferred concentration of TGF-jS to induce 
differentiation of cartilage repair cells may vary with 
the particular animal to be treated. 

It is necessary to stagger the exposure of the 
cartilage repair cells to the two concentration 
ranges of TGF-jS, since TGF-0 at relatively high 
concentrations (e.g., greater than 200 ng/ml of ma- 
trix solution) may not only transform cartilage repair 
cells into chondrocytes, but also will inhibit 
chemotactic attraction of cartilage repair cells; 
whereas at relatively low concentrations (e.g., 2-10 
ng/ml), TGF-0 attracts cartilage repair cells and 
stimulates their proliferation, but will not induce 
transformation of cartilage repair cells into chon- 
drocytes which produce cartilage tissue. 

In a preferred embodiment of this invention, in 
order to obtain the sequence of chemotaxis and 
proliferation, followed by transformation, TGF-/S is 
present both in a free, unencapsulated form and in 
an encapsulated, or otherwise sequestered, form in 
the matrix. Preferably, for the purpose of attracting 
and inducing proliferation of cartilage repair cells in 
the matrix and defect area, TGF-£ molecules are 
dissolved or suspended in the matrix at a con- 
centration of 2-10 ng/ml of matrix solution. To pro- 
mote transformation of cartilage repair cells in the 
matrix into chondrocytes, TGF-0 molecules are 
also present in the matrix sequestered in mul- 



tivesicular liposomes according to the method of 
Kim et al., 1983, supra, at a concentration of great- 
er than 200 ng/ml of matrix solution, and preferably 
at a concentration of greater than 500 ng/ml. The 

s TGF-0-loaded liposomes are disrupted when the 
attracted cartilage repair cells have populated the 
matrix and have started to degrade the matrix. 
During the degradation of the matrix, the cartilage 
repair cells ingest and/or degrade the liposomes, 

10 resulting in the release of TGF-0 at concentrations 
sufficient to induce the transformation of cartilage 
repair cells into chondrocytes. 

The required two-stage delivery of chemotactic 
and proliferating versus transforming concentra- 

75 tions of TGF-/8 may also be achieved by combining 
transforming concentrations of TGF-0 with a 
bioerodible polymer. Alternatively, a pump, and 
preferably an implanted osmotic pump, may be 
used to control the concentration of TGF-0 in the 

20 defect and matrix. In this embodiment of the inven- 
tion, the pump controls the concentration of TGF-/S 
in the matrix, i.e., the pump may release TGF-/S at 
an initial chemotactic and proliferation stimulating 
concentration and at a subsequent transforming 

25 concentration. Preferably, the transforming concen- 
tration of TGF-0 is delivered by the pump approxi- 
mately 1 to 2 weeks post-ope rati vely. Delivery of 
the transforming factor into the defect volume is 
preferably localized to the matrix in the defect site. 

30 The proliferation agents and, when used, the 
transforming factors in the compositions of this 
invention are applied in the defect site within the 
matrix. Their presence is thus restricted to a very 
localized site. This is done to avoid their free 

35 injection or infusion into a joint space. Such free 
infusion may produce the adverse effect of stimu- 
lating the cells of the synovial membrane to pro- 
duce joint effusion. 

In the compositions of this invention used in 

40 bone repair, one or more angiogenic factors is 
added to the matrix solution to stimulate the forma- 
tion and ingrowth of blood vessels and associated 
cells (e.g., endothelial, perivascular, mesenchymal 
and smooth muscle cells) and of basement mem- 

45 branes in the area of the bone defect. Angiogenic 
factors useful in the compositions and kits of this 
invention for stimulating vascularization throughout 
the deposited matrix in the area of the bone defect 
include bFGF, TGF-£, PDGF, TNF-a, angiogenin or 

so angiotropin. Heparin sulfate has been found to en- 
hance the angiogenic activity of bFGF. In a pre- 
ferred embodiment of this invention, bFGF and 
heparin sulfate are dissolved, suspended or bound 
in a matrix at a concentration of approximately 10 

55 ng/ml of matrix solution. The preferred concentra- 
tions for other angiogenic factors are: 5 ng/ml of 
matrix solution for TGF-jS, 10 ng/ml of matrix solu- 
tion for TNF-a, and 10 ng/ml of matrix solution for 



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PDGF. However, bFGF in combination with heparin 
sulfate is the most preferred angiogenic factor 
among the above named angiogenic factors. 

An osteogenic factor is also present in the 
matrix solution used in bone repair so that after 
blood vessels and associated cells have populated 
the matrix, the osteogenic factor is released into 
the bone defect site as the matrix is degraded in a 
concentration sufficient to promote a process lead- 
ing to the eventual development of osteoblasts and 
osteocytes. The osteogenic factor is sequestered 
or packaged in an appropriate delivery system 
within the matrix and is released as the matrix is 
degraded. The delivery systems used in the car- 
tilage repair compositions are useful in the bone 
repair compositions of this invention, e.g., lipo- 
somes or carbohydrate-based corpuscles (see 
supra). In one embodiment of this invention, the 
matrix used in bone repair contains TGF-0 pack- 
aged in a delivery system as the osteogenic factor, 
at a concentration of 100 ng/ml of matrix solution. 
Lower and higher concentrations of TGF-/5 may be 
used. 

Osteogenic factors useful in the bone repair 
compositions of this invention include any peptide, 
polypeptide, protein or any other compound or 
composition which induces differentiation of bone 
repair cells into bone cells, such as osteoblasts and 
osteocytes, which produce bone tissue. The 
osteogenic factors useful in this invention include 
proteins such as TGF-0 [Sampath, T. R. et al., J. 
Biol. Chem. , 265(22) , pp. 13198-13205 (1990)1 
osteogenin [Luyten, F. P. et al., J. Biol. Chem., 
264(15) , pp. 13377-80 (1989)], bone morphogenic 
protein (BMP) [Wang, E. et al., Proc. Natl. Acad. 
Sci. USA , 87, pp. 2220-24 (1990)], and TGF-0 
combined with epidermal growth factor (EGF). 

The differentiation of mesenchymal cells in- 
duced by an osteogenic factor may include the 
formation of intermediary tissues such as fibrous, 
hyaline and calcified cartilage; and endochondral 
ossification, which leads to the formation of woven 
bone tissue, which will become remodelled and 
transformed into mature lamellar bone tissue. In 
some instances, bone may be formed directly from 
mesenchymal cells without the appearance of an 
intermediary tissue. Within the matrix, the process 
of bone tissue formation usually occurs 3 to 4 
weeks after blood vessels have formed and infil- 
trated the matrix in response to the angiogenic 
factor present in the matrix. 

The matrix compositions described in this in- 
vention for repairing the bone portion of a full- 
thickness defect in joints are also useful in treating 
any defect in bone tissue as is desirable. Such 
defects include bone fractures, joint fractures, non- 
unions and delayed unions, percutaneous arth- 
rodesis, pseudo-arthrosis and bone defects result- 



ing from congenital defects, trauma, tumor infec- 
tion, degenerative disease and other causes of loss 
of skeletal tissue. The bone repairing matrix com- 
positions are also useful for prosthesis implantation 

5 and enhancement of prosthesis stability, enhance- 
ment of osseointegration of implant materials used 
for internal fixation procedures, stabilization of den- 
tal implant materials, healing acceleration of liga- 
ment insertion, and spine or other joint fusion pro- 

w cedures. 

Fibronectin or any other compound, including 
peptides as small as tetrapeptides, that contain the 
amino acid sequence Arg-Gly-Asp, may be used 
as cell adhesion promoting factors [Ruoslathi et al., 

is Cell, 44, pp. 517-18 (1986)] in order to enhance the 
initial adhesion of cartilage or bone repair cells to a 
matrix deposited in a defect site. Fibrin and certain 
collagen matrices already contain this sequence 
[Ruoslathi et al., 1986, supra]. When other biodeg- 

20 radable matrices are used, such cell adhesion pro- 
moting factors may be mixed with the matrix ma- 
terial before the matrix is used to fill or dress the 
defect. Peptides containing Arg-Gly-Asp may also 
be chemically coupled to the matrix material (e.g., 

25 to its fibers or meshes) or to a compound added to 
the matrix, such as albumin. 

The compositions hereinbefore described are 
useful in inducing cartilage or bone formation at a 
selected site of defect in cartilage or bone tissue of 

30 an animal. 

The kits and compositions of this invention 
allow for a treatment of cartilage and bone defects 
in animals, including humans, that is simple to 
administer and is restricted in location to an af- 

35 fected joint area. The entire treatment may be 
carried out by arthroscopic, open surgical or per- 
cutaneous procedures. 

In using the kits and compositions for treating 
defects or lesions in cartilage or bone according to 

40 this invention, a defect or lesion is identified, pre- 
pared, and filled with the matrix compositions ac- 
cording to this invention. 

In the case of repairing a defect in bone tissue, 
an angiogenic factor is present in the bone repair 

45 composition at an appropriate concentration to 
stimulate formation of blood vessels within the ma- 
trix filling the bone defect. As blood vessels are 
formed, the osteogenic factor is released from its 
delivery system to induce the process of bone 

so formation. 

For cartilage repair, a proliferation (mitogenic) 
agent is present in the matrix composition at an 
appropriate concentration to stimulate the prolifera- 
tion of cartilage repair cells in the matrix and defect 

55 or lesion. The same agent may also, at this con- 
centration, serve as a chemotactic agent to attract 
cartilage repair cells, provided that the factor used 
has a combined effect with respect to cell prolifera- 



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tion and chemotaxis (as does TGF-0 at 2-10 ng/ml 
of matrix). Alternatively, two different agents may 
be present in the matrix, one with a specific prolif- 
erative effect, and the other with a specific 
chemotactic effect. In an alternative embodiment, 
after the defect area is dressed with the matrix, the 
proliferation agent and, if desired, a chemotactic 
agent, may be injected directly into the matrix-filled 
defect area. 

In a subsequent step of cartilage repair, the 
cartilage repair cells in the matrix are exposed to a 
transforming factor at the appropriate time at a 
concentration sufficient to transform the cartilage 
repair cells into chondrocytes which produce stable 
cartilage tissue. This may be accomplished by 
including an appropriate delivery system containing 
the transforming factor within the matrix composi- 
tion as described above. Alternatively, the trans- 
forming agent may be delivered by injection di- 
rectly into the matrix-filled defect area at the appro- 
priate time. The transforming concentration should 
be made available to the cells approximately 1 to 2 
weeks following the initial implantation of the matrix 
into the defect area. Additional factors may be 
added to the delivery system or directly injected in 
order to better promote synthesis of the cartilage 
matrix components at this time point. 

Cartilage or bone defects in animals are readily 
identifiable visually during arthroscopic examination 
of the joint or during simple examination of the 
lesion or defect during open surgery. Cartilage or 
bone defects may also be identified inferentially by 
using computer aided tomography (CAT scanning) 
X-ray examination, magnetic resonance imaging 
(MRI) analysis of synovial fluid or serum markers, 
or by any other procedure known in the art. 

The kits and compositions of this invention 
may be used such that the bone defect site of a 
full-thickness defect is filled up to the calcified 
cartilage layer at the bone-cartilage interface with a 
bone repair matrix composition such that a flat 
plane is formed. Thereafter, the membrane of the 
kit, preferably a biodegradable membrane, which is 
impermeable to cells (e.g., pore sizes less than 5 
am), is placed over the matrix-filled bone defect, 
and the edges of the membrane sealed to the 
perimeter of the defect in the region of the 
cartilage-bone junction. Preferably, the membrane 
is sealed to the cartilage at the junction by thermal 
bonding using a thermal knife or laser. The matrix 
composition comprises a matrix material, an an- 
giogenic factor, and an osteogenic factor, which is 
packaged in an appropriate delivery system. 

The purpose of the membrane is to prevent 
blood vessels from infiltrating the layer of cartilage 
in the case of a full-thickness defect. The formation 
of blood vessels in the cartilage stimulates bone 
formation in the cartilage and inhibits complete 



repair of the cartilage layer. If only a bone defect 
needs to be repaired, no membrane has to be 
applied. 

After the membrane has been placed over the 

s matrix-filled bone defect and sealed to the perim- 
eter of the defect in the region of the cartilage- 
bone junction, the remaining portion of the defect is 
completely filled with a matrix composition used to 
stimulate cartilage repair. The composition for car- 

io tilage repair comprises a matrix material and a 
proliferation agent and, if desired, a chemotactic 
agent. The composition used in this step may also 
contain, packaged in an appropriate delivery sys- 
tem, a transforming factor. In the most preferred 

75 composition or kit for cartilage repair of the inven- 
tion, the matrix contains a proliferation agent, a 
chemotactic agent (which may be identical to the 
proliferation agent) and a transforming factor which 
is packaged in or associated with a delivery system 

20 that releases the transforming factor, at a time that 
the repair cells populating the matrix have begun 
remodelling the intercellular substance, at a con- 
centration that transforms the cartilage repair cells 
into chondrocytes. Preferred compositions are de- 

25 scribed above. 

The adhesion of a matrix to cartilage in a 
superficial defect or to the cartilage portion of a 
full-thickness defect can be enhanced by treating 
the cartilage defect with transglutaminase [see, 

30 e.g., Ichinose et al., J. Biol. Chem., 265 (3), pp. 
13411-14 (1990); Najjar, V. A. and Lorand, L, eds. 
Transglutaminases (Boston: Martinus-Nijhoff, 1984). 
In this embodiment of the invention, the cartilage 
defect is dried, e.g. by using cottonoid, and filled 

35 with a solution of transglutaminase. The solution is 
then removed, e.g., by suction, leaving a film con- 
taining transglutaminase on the cartilage. The de- 
fect is then filled with a matrix composition de- 
scribed above for cartilage repair. 

40 Additional details and examples describing kits 

and compositions for the treatment and repair of 
defects in cartilage are described in a commonly 
owned U.S. patent application, Serial No. 648,274, 
and are incorporated herein by reference. 

45 In order that the invention described herein 

may be more fully understood, the following exam- 
ples are set forth. It should be understood that 
these examples are for illustrative purposes and 
are not to be construed as limiting this invention in 

so any manner. 

EXAMPLE 

Repair Of Full-Thickness Defects In Articular Car- 
55 tilage 

Full-thickness articular cartilage defects, 0.7 
mm in width, were created in the medial condyles 



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and patellar grooves of adult mini-pig knee joints. 
Lesions were effected in a group of four animals 
maintained under general anaesthesia, using a 
planing instrument. The vertical extensions of each 
lesion into the subchondral bone (containing blood 
vessels and bone marrow cells) was controlled 
macroscopically by the occurrence of bleeding to 
insure that a full-thickness lesion had been made in 
the joint. The depth of the defect was filled in with 
a collagenous matrix, containing free TGF-0 at a 
concentration of about 4 ng/ml of matrix solution, 
and liposome-encapsulated TGF-0 at a concentra- 
tion of about 100 ng/ml of matrix volume. This 
osteogenic matrix composition was applied up to 
the cartilage-bone junction, at which level a cel- 
lulose membrane (pore size 0.2 urn), well adapted 
to the perimeter of the cartilage-bone junction of 
the defect area, was inserted. The remaining defect 
space was filled up to the surface level of the 
adjacent articular cartilage with a chondrogenic ma- 
trix composition as described in this application at 
page 15, lines 15-21; page 16, lines 7-11; and page 
22, lines 1-17. 

About ten weeks after the operation and treat- 
ment, the animals were killed and the knee joint 
components chemically fixed in buffered glutaral- 
dehyde (4%) solutions containing 2.5% Cetyl 
pyridinium chloride. Following dehydration, in a 
graded series of increasing ethanol concentration, 
and embedding in methylmethacrylate, histologic 
sections were produced and stained with McNeil 
Tetrachrome and Toluidine Blue O in preparation 
for light microscopic examination. 

That part of the defect space corresponding in 
level to the subchondral bone, i.e., where 
osteogenic matrix had been placed, was fully filled 
with newly-formed bone tissue. Likewise, the defect 
space adjacent to articular cartilage tissue, i.e., in 
the region above the cellulose membrane filled with 
the chondrogenic matrix composition, was filled 
with articular cartilage repair, tissue. 

Claims 

1. A composition for the treatment of defects in 
bone comprising: 

a matrix or matrix-forming material used to 
fill a defect in bone; 

an angiogenic factor at an appropriate con- 
centration to stimulate the formation and ing- 
rowth of blood vessels and associated cells in 
the matrix and the area of the defect; and 

an osteogenic factor associated with a de- 
livery system and at an appropriate concentra- 
tion such that upon delivery of the osteogenic 
factor to cells in the matrix and defect, the 
cells develop into bone cells which form bone. 



2. The composition according to claim 1 , wherein 
the angiogenic factor is selected from the 
group consisting of bFGF, a mixture of bFGF 
and heparin sulfate, TGF-0, PDGF, TNF-a, an- 

5 giogenin, angiotropin or combinations thereof. 

3. The composition according to claim 1 or 2, 
wherein the osteogenic factor is selected from 
the group consisting of TGF-£, a mixture of a 

70 TGF-0 and EGF, osteogenin, BMP and com- 

binations thereof. 

4. The composition according to any one of 
claims 1 to 3, wherein the matrix used to fill 

75 the defect area is selected from the group 

consisting of fibrin, collagen, gelatin, agarose, 
calcium phosphate containing compounds and 
combinations thereof. 

20 5. The composition according to any one of 
claims 1 to 3, wherein the angiogenic factor is 
bFGF present at a concentration of 5-10 ng/ml 
in the matrix and the osteogenic factor is TGF- 
0 associated with an appropriate delivery sys- 

25 tern which provides a concentration of TGF-/J 

of 100 ng/ml of matrix solution. 

6. The composition according to any one of 
claims 1 to 3, wherein the delivery system is 

30 selected from the group consisting of lipo- 

somes, bioerodible polymers, collagen fibers 
chemically linked to heparin sulfate prot- 
eoglycans, carbohydrate-based corpuscles, 
and water-oil emulsions. 

35 

7. A composition for the treatment of defects in 
bone comprising; 

a collagenous matrix solution; 

basic FGF present at a concentration of 5- 
40 10 ng/ml of matrix solution; and 

TGF-0 encapsulated in liposomes and 
present at a concentration of 100 ng/ml of 
matrix solution. 

45 8. The composition according to claims 1 to 7 for 
the induction of bone formation at a selected 
site in bone tissue of an animal wherein the 
composition is used to fill the site. 

so 9. A kit for treating full-thickness defects in joints 
in animals which comprises: 

a first matrix containing an effective 
amount of an angiogenic factor to stimulate 
55 formation and ingrowth of blood vessels with 

associated cells and containing an osteogenic 
factor associated with a delivery system that 
releases the osteogenic factor at a concentra- 



11 



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tion sufficient to induce differentiation of bone 
repair cells into bone cells which form bone, 
which is used for filling the bone portion of the 
full-thickness defect; 

a membrane which prevents migration of 5 
cells from the bone defect side to the cartilage 
defect side, which is used for covering the 
matrix-filled bone portion of the full-thickness 
defect and may be sealed to the perimeter of 
the defect in the region of the cartilage-bone k 
junction; and 

a second matrix containing an effective 
amount of a proliferation agent to stimulate 
proliferation of repair cells, an effective amount 75 
of a chemotactic agent to attract repair cells, 
and an effective amount of a transforming fac- 
tor associated with a delivery system that re- 
leases the transforming factor at a concentra- 
tion sufficient to transform repair cells into 20 
chondrocytes, which is used for filling the car- 
tilage portion of the full-thickness defect. 

10. The kit according to claim 9 further comprising 
transglutaminase which may be used for cov- 25 
ering the surface of the cartilage portion of the 
full-thickness defect prior to dressing the de- 
fect or lesion with the second matrix. 

11. The kit according to any one of claims 9 to 10 30 
wherein the osteogenic factor, the proliferation 
agent, and the transforming factor, are TGF-/3. 

12. The kit according to any one of claims 9 to 11 
in which the delivery system for the delivery of 35 
the transforming factor and the osteogenic fac- 
tor is selected from the group consisting of 
liposomes, bioerodible polymers, collagen fi- 
bers chemically linked to heparin sulfate prot- 
eoglycans, carbohydrate-based corpuscles, ao 
and water-oil emulsions. 



13. The kit according to any one of claims 9 to 12 
in which the first matrix is selected from the 
group consisting of fibrin, collagen, gelatin, 
agarose, and calcium phosphate containing 
compounds or combinations thereof. 



tion. 

16. The kit according to any one of claims 9 to 15 
wherein the angiogenic factor is bFGF present 
at a concentration of 5-10 ng/ml of the first 
matrix; 

the osteogenic factor is TGF-0 . encapsu- 
lated in liposomes and present at a concentra- 
tion of 100 ng/ml of the first biodegradable 
matrix; 

the proliferation agent and the chemotactic 
agent are TGF-0 present at a concentration of 
2-10 ng/ml of the second matrix; and 

the transforming factor is TGF-0 encap- 
sulated in liposomes and present at a con- 
centration of greater than 200 ng/ml of the 
second matrix. 

17. The kit according to any one of claims 9 to 16, 
wherein the first and second matrices further 
contain a cell adhesion promoting factor com- 
prising the tripeptide Arg-Gly-Asp. 

18. A kit for treating full-thickness defects in joints 
of animals which comprises: 

a first collagenous matrix containing bFGF 
present at a concentration of 5-10 ng/ml of the 
first collagenous matrix, and containing TGF-jS 
in liposomes at a concentration of 100 ng/ml of 
the first collagenous matrix, which may be 
used for filling the bone portion of the full- 
thickness defect; 

a membrane, which is impermeable to 
blood vessels and cells, which may be used 
for covering the first collagenous matrix-filled 
bone portion of the full-thickness defect and 
may be sealed at its perimeter to the edges of 
the defect in the region of the cartilage-bone 
junction; 

a second collagenous matrix containing 
TGF-j8 at a concentration of 2-10 ng/ml of the 
second collagenous matrix, and containing 
45 TGF-0 in liposomes at a concentration of 

greater than 200 ng/ml of the second col- 
lagenous matrix, which is used for filling the 
cartilage portion of the full-thickness defect. 

19. The kit according to claim 18 further compris- 
ing the step of covering the surface of the 
cartilage portion of the full-thickness defect 
with transglutaminase prior to filling the defect 
with the second collagenous matrix. 

20. The composition according to claim 5 or 7 
further comprising an amount of heparin sul- 
fate sufficient to enhance the angiogenic activ- 



14. The kit according to any one of claims 9 to 13 
in which the second matrix is selected from 50 
the group consisting of fibrin, collagen, gelatin, 
agarose, and combinations thereof. 

15. The kit according to any one of claims 9 to 14, 
wherein the first and the second matrix is fibrin 56 
which is formed by addition of thrombin to a 
solution of fibrinogen immediately before filling 
the defect or lesion with the fibrinogen solu- 



12 



23 



EP 0 530 804 A1 



24 



ity of the bFGF. 

21. The kit according to claim 16 or 18 wherein 
the first matrix also contains an amount of 
heparin sulfate sufficient to enhance the an- 
giogenic activity of the bFGF. 

22. The kit according to claim 11 wherein the 
angiogenic factor is bFGF. 

23. The use of the composition in any one of 
claims 1-8 to prepare a medicament for the 
treatment of defects in bone. 

24. A method of preparing a composition for the 
treatment of defects in bone comprising: 

adding to a matrix or matrix-forming ma- 
terial useful for filling a defect in bone an 
amount of an angiogenic factor sufficient to 
stimulate the formation and ingrowth of blood 
vessels and associated cells in the matrix and 
area of the bone defect, and an amount of an 
osteogenic factor associated with an appro- 
priate delivery system which can provide a 
sufficient concentration of osteogenic factor to 
stimulate the formation of bone cells. 

25. A method for making a kit for treating full- 
thickness defects in joints in animals compris- 
ing: 

combining a first matrix or matrix-forming 
material useful to fill a defect in a bone with an 
angiogenic factor at a concentration sufficient 
to stimulate the formation and ingrowth of 
blood vessels and associated cells in the ma- 
trix and area of the bone defect and an 
osteogenic factor associated with an appro- 
priate delivery system which can provide a 
sufficient concentration of osteogenic factor to 
blood vessels and associated cells in the ma- 
trix and defect, to stimulate the formation of 
bone ceils; 

providing a membrane which can be seal- 
ed to the perimeter of the cartilage-bone junc- 
tion of the full-thickness defect to cover the 
matrix-filled bone portion of the full-thickness 
defect and prevent the migration of cells from 
the bone defect side to the cartilage defect 
side of the full-thickness defect; and 



tion of the full-thickness defect, and a 
chemotactic agent at a sufficient concentration 
to attract repair cells to the matrix in the car- 
tilage portion of the full-thickness, and a trans- 

5 forming factor associated with an appropriate 

delivery system which can provide a sufficient 
concentration of the transforming factor to 
stimulate the formation of cartilage from the 
repair cells in the matrix-filled cartilage portion 

70 of the full-thickness defect. 



75 



20 



25 



30 



35 



40 



45 



50 



combining a second matrix or matrix-for- 
ming material useful to fill the cartilage portion 55 
of the full-thickness defect with a proliferation 
agent at a sufficient concentration to stimulate 
proliferation of repair cells in the cartilage por- 



13 



J 



European Patent 
Office 



EUROPEAN SEARCH REPORT 



Application Number 



EP 92 11 5079 



DOCUMENTS CONSIDERED TO BE RELEVANT 




CMetory 


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


Relevant 
to claim 


CLASSIFICATION OF THE 
APPLICATION (Int. CI.S) 


X 


WO-A-9 005 755 (COLLAGEN CORP.) 
* Page 11, lines 12-34; claims * 


1-25 


A 61 L 25/00 
A 61 L 27/00 


X 


EP-A-0 308 238 (ETHICON INC.) 
* Page 3, lines 49-55; claims * 


1-25 


A 61 K 37/02 


X 


EP-A-0 361 896 (COLLAGEN CORP.) 
* Claims * 


1-4,6,8 
,10 




X 


EP-A-0 375 127 (GENENTECH, INC. ) 

* Column 10, lines 28-42; examples 1-3 


1-4,6,8 
,10 




X 


ANNALS OF SURGERY, vol. 211, no. 3, 
March 1990, pages 288-294, 
Philadelphia, PA, US; G.A. KSANDER et 
al.: "Exogenous transforming growth 
factor-beta 2 enhances connective 
tissue formation and wound strength in 
guinea pig dermal wounds healing by 


1-4,6,8 
,10 






secondary intent" 

* Abstract; discussion * 




TECHNICAL FIELDS 
SEARCHED (lot. CI.S) 


X 


WO-A-9 009 783 (MASSACHUSETTS 
INSTITUTE OF TECHNOLOGY) 
* Page 5, lines 25-31; page 6, lines 
1-31; page 7, lines 1-5 * 


1-4 


A 61 L 

A 61 K 


D,P 
X 


WO-A-9 213 565 (R.F. SHAW) 
* Whole document * 


1-25 




The present search report has been drawn op for all claims 







Place of search 

THE HAGUE 



Date of coaqdctloo of the learck 

09-12-1992 



ESPINOSA Y CARRETERO M 



CATEGORY OF CITED DOCUMENTS 



X : particularly relevant if tarn en alone 

V : particularly relevant if combined witb another 

document of the same category 
A : technological background 
O : DOD-writteo disclosure 
P : Intermediate document 



T : theory or principle underlying the Invention 
E : earlier patent document, but published on, or 

after the filing date 
D : document died in the application 
L : document cited for other reasons 



A : member of the same patent family, corresponding 
document 



J 



European Patent 
Office 



EUROPEAN SEARCH REPORT 



Page 2 

Application Number 

EP 92 11 5079 



DOCUMENTS CONSIDERED TO BE RELEVANT 



Category 



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



Relevant 
to i 



CLASSIFICATION OF THE 
APPLICATION ant CI. 5) 



WO-A-8 904 646 (S.R. JEFFERIES) 
* Examples 2-3 * 

WO-A-8 600 526 (A.I. CAPLAN) 



1-10 



TECHNICAL FIELDS 
SEARCHED 0nl. C1.5) 



The present search report has been drawn up for all claims 



Place of Hwcb 

THE HAGUE 



D*e of cooptetfcw of ti* tetrch 

09-12-1992 



ESPINOSA Y CARRETERO M 



CATEGORY OF CITED DOCUMENTS 

X : particularly relevant if taken alone 

Y : particularly relevant if combined wltb another 

document of the same category 
A : tech do logical background 
O : n on- written disclosure 
P : Intermediate document 



T : theory or principle underlying the Inventioo 
E : earlier patent document, but published on, or 

after the filing date 
D : document dted in the application 
L : document cited for other reasons 

i : member of the same patent family, corresponding 
document 



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