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WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




PCX 

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (POT) 



(51) International Patent Classification ^ : 
C12N 5/00, A61L 27/00 



Al 



(11) international Publication Number: WO 97/45532 

(43) International Publication Date: 4 C^ember 1997 (04.12.97) 



(21) International Application Number: PCT/US97/O9067 

(22) International Filing Date: 28 May 1997 (28.05.97) 



(30) Priority Data: 

60/018,492 



28 May 1996 (28.05.96) 



US 



(71) Applicant: BROWN UNIVERSITY RESEARCH FOUNDA- 

TION [US/US]; 42 Charksncld Street, P.O. Box 1949, Prov- 
idence, Rl 02912 (US). 

(72) Inventors: VALENTINI. Robert, F.; 28 Selkirk Road. 

Cranston, Rl 02905 (US). KIM. Hyun, D.; Apartment 4A. 
140 Prospect Street, Providence, Rl 02906 (US). 

(74) Agent: KINDREGAN, Helen; Wolf, Greenfield & Sacks. PC, 
600 Atlantic Avenue, Boston, MA 02210 (US). 



(81) Designated Stales: CA, JP. European patent (AT, BE, CH, DE, 
DK, ES. FI, FR, <5B, GR, IE, IT, LU. MC. NL, PT, SE). 



Published 

With international search report. 

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



(54) Title: HYALURONAN BASED BIODEGRADABLE SCAFFOLDS FOR TISSUE REPAIR 
(57) Abstract 



A hyaluronic acid derivitized scaffold and method of forming arc disclosed. The scaffolds are useful for various medical purposes 
such as tissue repair, tissue reconstruction and wound healing. In order to enhance these processes the scaffolds may be engineered to 
tncoiporate biologically active molecules such as BMP. 



FOK THE PURPOSES OF INFORMATION ONLY 
codes used to identify Sutes party .o Ae PCT on .he froa. pages of pamphlet publishing international applications under the PCT. 



AL 


Albania 


AM 


Aimenia 


AT 


Austria 


AV 


Ausiralia 


AZ 


Azeibaiian 


BA 


Bosnia and Heraegovina 


BE 


Barbados 


BE 


Belgium 


BF 


Burkina Faso 


BG 


Bulgaria 


BJ 


Benin 


BR 


Brazil 


BY 


Belanis 


CA 


Canada 


CF 


Central African Reinrt>lic 


CG 


Congo 


CH 


Switzerland 


CI 


Cd« d'lvoiit 


CM 


Cameroon 


CN 


China 


cu 


Cuba 


cz 


Cxech Republic 


DE 


Germany 


DK 


Denmark 


EE 


Estonia 



ES 

Ft 

FR 

GA 

GB 

GE 

GH 

GN 

GR 

HV 

IE 

It 

IS 

IT 

JP 

K£ 

KG 

KP 

KR 
KZ 
LC 
LI 
LK 
LR 



Spain 
Finland 
France 
Gabon 

Unhcd Kingdom 

Georgia 

Ghana 

Guinea 

Greece 

Hungary 

Ireland 

Israel 

Iceland 

Italy 



Kenya 
Kyrgyzstan 
Democratic Pc<^Ie*5 
Republic of Korea 
Republic of Korea 
Kazak&tan 
Saint tucia 
Liechteitatcin 
Sri l^ka 
Liberia 



L5 

LT 

LU 

LV 

MC 

MD 

MG 

MK 

ML 
MN 
MR 
MW 
MX 
NE 
. NL 
NO 
NZ 
PL 
PT 
RO 
RU 
SO 
SE 
SG 



Lesotho 

Lithuania 

lAiKcmbourg 

Ijuvia 

Monaco 

Republic of Moldova 

Madagascar 

The foimer Yugoslav 

Republic of Macedonia 

Mali 

Mongolia 

Mauritania 

Malawi 

Mexico 

Niger 

. Netherlands 
Norway 
New Zealand 
Poland 
Portugal 
Romania 

Ruuian Federation 
Sudan 
Sweden 
Singapore 



SI 


Slovenia 


SK 


Slovakia 


SN 


Senegal 


sz 


Swaziland 


TD 


<n»ad 


TG 


Togo 


TJ 


Tajikisian 


TM 


Turionenistan 


TR 


Turkey 


TT 


Trinidad and Tobago 


UA 


Ukraine 


UG 


Uganda 


US 


United States of America 


HZ 


Uzbekistan 


VN 


Viet Nam 


YD 


Yugoslavia 


ZW 


Zimbabwe 



wo 97/45532 PCT/US97/09067 
HVAMIRONAN BASED BIODEGRADABLE SCAFFOLDS FOR TISSUE REPAIR 



Background Of The Invention 

5 Hyaluronic acid is a naturally occurring mucopolysaccharide consisting of alternating 

glucuronic acid and N-acetyl D-glucosamine monomers. It is present in connective tissues and 
plays a vital role in many biological processes such as tissue hydration, proteoglycan 
organization in the extra-cellular matrix, and cell differentiation. Because of its important 
biological roles, hyaluronic acid has been widely exploited in medical practice for use in treating 

10 many different conditions. Several hyaluronic acid containing products are currently marketed 
for pharmaceutical or veterinary use, including a product for intra-ocular injection during eye 
surgery, synovitis agents for veterinary use, and coated gauzes for wound dressings. 

Through the esterification of carboxyl groups of hyaluronic acid with various 
therapeutically inactive and active alcohols, it has been possible to synthesize biopolymers with 

15 medically desirable properties that are significantly different from those of hyaluronic acid itself. 
The biocompatibility of these altered molecules appears to be quite good. For example, a 
chemically modified form of hyaluronic acid "HYAFF-1 1" prevents fast enzymatic degradation 
in vivo and degrades slowly in concert with new tissue formation. The HYAFF -1 1 material is 
commercially available as skin repair products for wound and bum patients, 

20 The following depicts the structure of hyaluronic acid. 



25 



H i r^ OH 




The carboxyl group of hyaluronic acid can be replaced with various moieties, including 
30 molecules such as ethyl, propyl, pentyl, benzyl or larger molecules such as hydrocortisone or 
methyl prednisolone. Reaction conditions can be controlled so as to influence the degree of 
substitution on the hyaluronic acid molecule, thereby further influencing the properties of the 



wo 97/45532 



-2- 



PCT/US97/09067 



final product. 

Summary n f the Invention 

The invention utilizes derivatives of hyaluronic acid as raw material to fabricate porous, 
5 degradable scaffolds for a variety of medical purposes, including, but not limited to, tissue repair 
and reconstruction and wound healing. These scaffolds are biocompatible and have degradation 
products that are substantially non-toxic. These products are an improvement oyer prior art 
materials such as poly lactide, poly glycolide, their copolymers, and the like which, although 
biocompatible, render acidic degradation products that are not necessarily optimal for tissue 
10 repair. 

The porous scaffolds of tiie invention can be fabricated to any size or shape and can be 
produced to virtually any desired predetermined pore size, depending upon the application. The 
scaffolds of the invention can be adapted to promote host cells of different varieties to migrate, 
adhere, proliferate, differentiate, and synthesize new tissue inside the pores. The invention can 
1 5 accelerate the infiltration and integration of host tissue, while degrading slowly in concert with 
new tissue formation. In addition, the porous scaffold can be used as a substrate for covalent or 
non-covalent attachment of bioactive molecules such as cytokines, peptides, proteins, etc. that 
have specific effects on ingrowing cells or surrounding tissue. Depending upon the bioactive 
molecules selected, these effects on ingrowing cells can be directed to enhancing cell migration, 
20 adhesion, commitment, proliferation and/or differentiation. 

According to one aspect of the invention, a method for forming a substrate for cell growth 
is provided. A water-insoluble hyaluronic acid derivative is dissolved in a first solvent. A 
mixture of the first solvent, the water-insoluble hyaluronic acid derivative and a pore forming 
agent is formed, wherein the pore forming agent is insoluble in the first solvent. The mixture 
25 then is contacted with a second solvent, wherein the water-insoluble hyaluronic acid derivative is 
insoluble in the second solvent, but the pore forming agent is soluble in the second solvent, 
whereby the first solvent and the pore forming agent are extracted from the mixture to produce a 
porous scaffold of the water-insoluble hyaluronic acid derivative. 

The pore forming agent preferably is sized so as to leave pores sufficient to permit cell 
30 ingrowth into the scaffold when the pore forming agent is extracted fi-om the mixture. The 
resulting scaffold is a three-dimensional structure of interconnected pores which permit cell 
ingrowth and, eventually, tissue replacement of the scaffold. In one particularly preferred 



wo 97/45532 PCT/US97y09067 

- 3 - 

embodiment, the pore forming agent is particles having a diameter between 10-1000 micrometers 
with optimal tissue ingrowth at 1 06 and 600 micrometers. 

It further is preferred to vacuum dry the scaffold from a wet state at a temperature of 
between 4°C and 30°C, most preferably at ambient or room temperature. This results in a 
5 scaffold that is non.brittle, handleable, and capable of being auloclaved with steam or gas 
without undesirably affecting the pore interconnectivity of the scaffold. Most preferably the 
water-insoluble hyaluronic acid derivative is hyaluronic acid esterified with a benzyl moiety. 

In further embodiments, the method involves agents which are attached to the scaffold. 
The attachment may be covalent or non-covalent attachment. The attachment may be directly to 
10 the hyaluronic acid derivative in advance of the scaffold formation, or may be applied covalently 
or non-covalently after the formation of the scaffold, such as by a coating. The agent also may 
be blended with the dissolved hyaluronic acid derivative in the first solvent, to cause the agent to 
be intermixed with and part of the formed scaffold structure. The kinds of agents contemplated 
for attachment to the scaffold include drug agents for being released to surrounding tissues, 
15 antipathogens for inhibiting pathogenic invasion of the scaffold, cell stimulating agents for 

causing, for example, cell migration, adhesion, commitment, proliferation and/or differentiation 
in, on or within the scaffold, and bioerodable coatings or blending agents for influencing the 
bioerodability of the scaffold and/or for containing any of the foregoing agents such as 
antipathogen agents, drug agents or cell stimulating agents. 
20 According to another aspect of the invention, a substrate for cell growth is provided. The 

substrate is a scaffold of vvater-insoluble derivatized hyaluronic acid defining interconnected 
pores of sufficient size to permit mammalian cell ingrowth into the pores, wherein the 
derivatized hyaluronic acid is a covalent conjugate of hyaluronic acid and a water-insoluble 
moiety that renders the conjugate insoluble in water. The preferred features of the scaffold are as 
25 described above in connection v^th the methods. All the various products resulting from the 
foregoing methods are intended to be embraced by this aspect of the invention. For example, 
scaffolds coated non-covalently or covalently with bioactive agents are contemplated by the 
invention. Likewise, scaffolds coated with or blended with bioerodable polymers are 
contemplated by the invention. The preferred covalent conjugate of hyaluronic acid is esterified 
30 with benzyl moieties, most preferably wherein 100% of the carboxyl moieties of the hyaluronic 
acid are esterified with benzyl moieties. Scaffolds composed of hyaluronic acid esterified with 
other moieties (e.g. drugs, peptides) may also be employed. 



wo 97/45532 PCT/US97/09067 

-4- 

According to another aspect of the invention, a two-phase scaffold is provided. The iv/o: 
phase scaffold is prepared by adding a hydrogel, a biodegradable polymer such as poly lactic acid 
(PLA) or polyglycolic acid (PGA), or a ceramic such as hydroxyapatite or iricalcium phosphate 
to a hyaluronic acid solution. Preferably the hyaluronic acid solution is HYAFF and the two- 
phase scaffold is prepared with a hydrogel The pores of the two-phase scaffold are filled with 
the hydrogel. The two-phase scaffold has all of the preferred features as described above in 
connection with the single-phase scaffold of the invention. 

According to another aspect of the invention, methods for growing cells are provided. 
These cells are contacted with the scaffold of the invention as outlined above, and permitted to 
grow upon and/or into the pores of the scaffold. 

According to another aspect of the invention, methods of stimulating cell differentiation 
are provided by contacting cells with the scaffold of the invention. 

According to another aspect of the invention, methods for tissue culture are provided by 
contacting cells with the scaffold of the invention. 

According to still other aspects of the invention, methods for treating a variety of 
conditions are provided, including methods for reconstituting or repairing bone, methods for 
accelerating wound healing, methods for repairing cartilage as well as methods for reconstituting 
tissues in the ectodermal, mesodermal and endodermal layers that require replacement or 
regeneration. 

Scaffolds with cells seeded upon the scaffolds also represent an aspect of the invention. 
One particularly important aspect of the invention is the stimulation of bone cells isuch as 
osteoblasts and pre-osteoblasts, and precursors thereof, to grow within a scaffold which can be 
precoated, dipped or filled vnih bone morphogenetic proteins in order to induce bone growth and 
differentiation from bone precursor cells. 

These and other aspects of the invention will be described in greater detail below. 

firief Descript ion Of The Drawings 

Figure 1 is a graph depicting the release of BMP from Hyaluronic Acid scaffolds, 
prepared as a scaffold alone (HA S), as a scaffold precoated with BMP and dried (HA P), as a 
scaffold in which BMP and collagen are gelled together (HA SCB), and a scaffold in which the 
scaffold is precoated with BMP and then collagen (HA PC); and 

Figure 2 is a graph depicting the release of BMP from Poly-lactic Acid scaffolds, 



wo 97/45532 PCT/US97/0!H)67 

- 5 - 

prepared as a scaffold alone (PLA S), as a scaffold precoated with BMP and dried (PLA P), as a _ 
scaffold in which BMP and collagen are gelled together (PLA SCB), and a scaffold in which the 
scaffold is precoated with BMP and then collagen (PLA PC) 

5 Detailed Description Of T he Invention 

The invention involves three-dimensional biodegradable scaffolds of hyaluronic acid 
derivatives for tissue reconstruction and repair. The porous scaffold has interconnected pores 
that permit cells to grow into the scaffold, preferably completely penetrating the scaffold with 
cells, and thereby, eventually replacing the scaffold with tissue. The scaffold can be fabricated to 

10 be virtually any shape, size or thickness, and can be produced to various porosities and pore 
sizes, depending upon the application. The scaffold is degradable, so that eventually it can be 
completely replaced by tissue. The scaffold degrades slowly in concert with new tissue 
formation. Such a scaffold offers the advantage of promoting host cells to migrate, adhere, 
proliferate and synthesize new tissue inside the pores, accelerating, for example, wound healing. 

15 Void volumes for the scaffold according to the invention can range from 40-90%. Pore 

sizes for the scaffold of the invention can range from 10-1000 micrometers. 

The invention requires the use of hyaluronic acid derivatives that are water-insoluble, but 
are soluble in a first solvent. The water-insoluble hyaluronic acid is dissolved in that first 
solvent, together with a pore forming agent that is insoluble in the first solvent. That mixture 

20 then is contacted with a second solvent in which the hyaluronic acid derivative is insoluble but in 
which the pore forming agent is soluble. In this manner, the first solvent is replaced/extracted by 
the second solvent in which the hyaluronic acid is insoluble, bringing the hyaluronic acid 
derivative out of solution and forming a scaffold. Likewise, the pore forming agent is soluble in 
the second solvent and is extracted/dissolved, thereby leaving a porous scaffold of the water- 

25 insoluble hyaluronic acid derivative. 

The water-insoluble hyaluronic acid derivatives are known to those skilled in the art and 
described in numerous publications. For example, because hyaluronic acid is a polycarboxylic 
acid, its water-insoluble esters may be prepared using standard methods for the esterification of 
carboxylic acids, such as the treatment of free hyaluronic acid with the desired water-insoluble 

3D moieties in the presence of appropriate catalysts. Alternatively, the esters may be prepared by 
treating a quaternary ammonium salt of hyaluronic acid with an esterifying agent in a suitable 
aprotic solvent. Details of this latter method have been described in European Patent Application 



wo 97/45532 PCTAJS97/09067 

-6- 

No. EP216453, April 1, 1987, the disclosure of which is incorporated herein by reference. 
Esterification of hyaluronic acid with suitable water-insoluble moieties may be achieved also by 
the use of linking groups interposed between the hyaluronic acid and the water-insoluble moiety. 
Likewise, hyaluronic acid may be derivatized via amide bonds, as will be clear to those 
5 skilled in the art. Such hyaluronic acid derivatives are described in the following PCT 

publications, the disclosure of which is incoiporated herein by reference. W095/24429 discloses 
highly reactive esters of carboxy polysaccharides, including hyaluronic acid. PCT Patent 
applications W095/24497 and W095/041 32 disclose methods for preparing high molecular 
weight hyaluronic acid derivatives. 
10 Hyaluronic acid is a linear polysaccharide. Many of its biological effects are a 

consequence of its ability to bind water, in that up to 500 ml of water may associate with 1 gram 
of hyaluronic acid. Esterification of hyaluronic acid with uncharged organic moieties reduces the 
aqueous solubility. Complete esterification with organic alcohols such as benzyl renders the 
hyaluronic acid derivatives virtually insoluble in water, these compounds then being soluble only 
15 in certain aprotic solvents. 

When films of hyaluronic acid are made, the films essentially are gels which hydrate and 
expand in the presence of water (hydrogels). By esterifying the hyaluronic acid and making it 
insoluble in water, the scaffolds of the present invention then are possible. The scaffolds are not 
hydrated in the presence of water and maintain their overall structure, permitting cell ingrowth. 
20 Thus, the hyaluronic acid derivatives useful according to the invention are those sufficiently 
derivatized such that the hyaluronic acid derivative will not form a hydrogel. Those of ordinary 
skill in the art can easily test whether sufficient derivitization with an uncharged moiety has 
occurred so as prevent the formation of a hydrogel. The preferred hyaluronic derivative is 100% 
esterified hyaluronic acid-benzyl covalent conjugates, sold under the trade name HYAFF by 
25 Fidia Advanced Biopolymers, Abano Terme, Italy. 

Solvents for the water-insoluble derivatized hyaluronic acid molecules include 
dimethylsulfoxide (DMSO), N-methyl-pyrrolidone (NMP), 1, 1. 1, 3, 3, 3-hexafluoro-2-propanol 
(HFIP) and dimethylacetamide (DMAC). Other appropriate solvents will be known to those of 
ordinary skill in the art. NMP is the prefened solvent. 
30 Non-solvents for the derivatized hyaluronic acid useful in the invention include water, 

ethanol, isopropanol, glycerol, ethyl acetate, tetrahydrofuran, and acetone. Other non-solvents 
will readily be known to those of ordinary skill in the art: To be clear, the non-solv«nt ("second 



wo 97/45532 PCT/US97/09067 

-7- 

solvent") is used to replace the solvent and cause the extraction of the first solvent such as NMP_ 
or DMSO, thereby causing the formation of the scaffold and to dissolve the pore forming agent, 
thereby producing pores in the scaffold. 

The pore forming agents usefiji in the invention are particles of a desired size that arc 
insoluble in the first solvent but that are soluble in the second solvent The particles preferably 
are sized and are present in sufficient concentration so as to create pores of a sufficient size to 
permit a plurality of mammalian cells to grow into and throughout the interconnected pores. In 
one particularly preferred embodiment involving bone grow^th, the particles are between 1 00 and 
600 micrometers in diameter. The pore forming agents may be any of a variety of materials, 
depending on the particular selection of the solvent and non-solvent. Examples include: salt 
crystals such as NaCl, KCL, MgClj, CaCl. and BaS04; soluble proteins such as albumin, 
globulins, and the like; soluble dextrans such as dextran and dextransulfates, and the like; soluble 
hydrogels such as agarose, alginate, chitosan, cellulose, carboxymethylcellulose, and the like; 
and microspheres of polylactic acid, polyglycolic acid, and the like. Those of ordinary skill in 
the art will readily be able to select usefiil pore forming agents. Tables 1 , 2, and 3 in the 
Examples provide examples of the use of different sizes and concentrations of NaCl as well as 
various lyophilization techniques to produce a variety of pore sizes and shapes. 

As mentioned above, the scaffolds may be coated with a variety of materials, including 
bioactive agents, and bioerodable agents. Bioactive agents include antipathogenic agents such as 
antibiotics, antivirals, and antifungals, antiinflammatory agents, immunomodulators, cytokines, 
etc. Virtually any bioactive compound useful in the scaffold or in the environment of the 
scaffold may be coated onto the scaffold. In one particularly important embodiment, bioactive 
molecules that have specific effects on ingrowing cells are coated onto the scaffold. Such 
molecules can be those that effect cell migration, cell adhesion, cell conunitment, cell - 
proliferation, cell differentiation, etc. Such molecules include interlukins, interferons, bone 
morphogenetic factors, growth factors including platelet-derived growth factor, epidermal 
growth factor, transforming growth factor and fibroblast growth factor and colony stimulating 
factors. In one important aspect of the invention, the scaffold is coated with bone morphogenetic 
proteins (BMPs) or growth and differentiation factors (GDFs) in order to induce the formation of 
differentiated bone cells from bone precursor cells. 

The coating also can be a biodegradable polymer which is added to influence the 
degradation rate of the scaffold. Biodegradable polymers useful according to the invention 



wo 97/45532 PCTAJS97/09067 

-8- 

include polylactic acid, polyglycolic acid, polylactic-polyglycolic copolymers, polycaprolactone, 
polyphosphazenes and polyorthesters. Other biodegradable polymers are well known to those of 
ordinary skill in the art and are described in great detail in the art relating to tissue implants and 
sustained release polymeric devices. 

Instead of coating the scaffold with the foregoing polymer materials a two-phase scaffold 
may be prepared, in which the scaffold pores may be filled with the foregoing materials or a 
hydrogel or ceramic. The two-phase scaffold may be prepared as described below in Example 5. 
The two-phase scaffold has all of the preferred features as described above in connection with the 
single-phase scaffold of the invention. 

As mentioned above, the materials may be non-covalently coated on the scaffolds or 
covalently attached to the scaffolds. If covalently attached to the scaffolds, such covalem 
attachment may be carried out prior to the formation of the scaffold or may be carried out after 
formation of the scaffold. Drugs may be incorporated in a gel which solidifies within the 

scaffold (e.g. collagen type I). 

Many of the objects and advantages described above in connection with coatings may be 
achieved by blending such materials in solution with the water-insoluble hyaluronic acid 
derivative prior to formation of the scaffold. For example, biodegradable polymers may be 
included in such solutions, with the resulting scaffold being a blend of the derivatized hyaluronic 
acid and the biodegradable polymer. Likewise, bioactive molecules may be blended with the 
water-insoluble hyaluronic acid derivative prior to formation of the scaffolding. When a 
biodegradable polymer is blended with the hyaluronic acid derivatives of the invention, then it is 
prefeired that the biodegradable polymer comprise less than 50% of the total material of the 
scaffold, and more preferably 10% or less of the total material of the scaffold. In any event, the 
nature of the biodegradable polymer and amount must be adjusted, depending upon the 
hyaluronic acid derivative selected and the desired characteristics of the end product so that the 
final scaffold has the characteristics desired for the particular application. The biodegradable 
polymer of this embodimem, however, should be chosen so that it is not dissolved by the water 
or other non-solvent. If the biodegradeable polymer is soluble, it may be chemically modified to 
make it insoluble. Techniques for chemical modification are well known to those of skill in the 
3 art. 

It is preferred that the scaffold be dried from the wet state by lyophilization without 
freezing. In other words, a vacuum pressure is applied to dry the scaffold. It is prefened that the 



wo 97/45532 PCT/US97/09067 

.9- 

vacuum pressure be applied at about ambient or room temperature, because doing so at either an_ 
elevated temperature or by freeze-drying adversely affects the interconnectivity of the pores and 
the overall structure of the scaffold. According to the methods of the invention, scaffolds are 
produced with not only desirable porosity for cell ingrowth, but also with a structural integrity so 
that they may be sterilized using steam or gas sterilization without adversely affecting the 
scaffold structure and characteristics. 

The scaffolds of the invention have a variety of clinical uses. One important ex^ple is 
in the repair of bone defects caused by trauma, bone tumor resection, in the case of joint fusion 
and spinal fusion for non-healing fractures and osteoporotic lesions. It is noted that the scaffold 
may be seeded with bone cells (osteoblasts and osteocytes) and bone cell precursors 
(mesenchymal stem cells from bone marrow, periosteum, endosteum, etc.) before implantation. 
The scaffolds also may be used in treating tooth and jaw defects in cases of trauma, bone loss, 
tooth loss, gum disease and the like. The scaffold again can be seeded with cells of the foregoing 
type for such purposes. The scaffolds also are useful in treating cartilage defects such as those 
which result from rheumatoid arthritis, osteoarthritis and trauma. Cells useful for seeding in 
such circumstances are chondroblasts and chondrocytes and cartilage cell precursors such as the 
cell precursors described above in connection with bone. The scaffolds also may be used to 
repair defects and damage in skin, muscle and other soft tissues such as results from trauma, 
bums, ulcers (diabetic ulcers, pressure sores, venus, stasis ulcers, etc.). In this case, scaffolds can 
be seeded with, for example, dermal fibroblasts, keratinocytes, and skeletal muscle cells. 
Likewise, damage to visceral organs including liver damage, heart attack damage, and damage 
resulting from intestinal cancer or intestinal ulcer may be treated with the scaffolds of the 
invention. In these instances, the scaffolds can be seeded with cells such as hepatocytes, cardiac 
muscle cells, intestinal cells, etc. 

The invention also pertains to in vitro culture of cells with the purpose of creating tissue 
constructs for repairing tissues and organs in vivo. The scaffolds may be used to promote tissue 
culture of committed cells and/or differentiation of precursor cells. Thus, the scaffolds of the 
invention can be used in virtually all instances when it is desirable to provide a substrate for the 
growth of cells onto or into a tissue replaceable matrix. Scaffolds can also be used with 
autografts, allografts, and xenografts associated with bone grafts, cartilage grafts, and joint 
resurfacing implants and are particularly important applications of the present invention. 



wo 97/45532 



PCTAJS97/09067 



- 10- 



F.Tgmples 

F^ample 1 : Pry pamtion of Polvmer ScaffoWs- 

/. Scaffold preparation: 

n\ 1 Scaffold Fahrication Techniqw: 

HYAFF-1 1, a 100 % esterified derivative of hyaluronic acid (commercially available 
from Fidia Advanced Biopolymers, Abano Terme, Italy) was dissolved in N-melhyl pyrrolidone 
for 12 hours at room temperature to make a 1 0 % (w/v) solution. Presieved NaCl crystals (Fisher 
Scientific, or Sigma) were mixed for 1 0 minutes at 20°C with the polymer solution at salt to 
polymer dry weight ratio of 9:1 or 1 5:1 (w/w) to create a slurry or a paste-like mixture. The size 
and quantity of NaCI used determines the porosity, pore distribution, and interconnectivity of the 
final scaffold product. The quantity used in each experiment, therefore, was adjusted in order to 
produce a scaffold having the desired porosity, pore distribution, and interconnectivity. The 
average porosity, pore distribution, and interconnectivity produced at particular concentrations of 
NaCI is depicted in Table 1 . The final scaffold pore size is dependent on the size of the NaCl 
crystals used and, therefore, the size of the NaCl crystals to be used was determined based on the 
desired pore size. The desired size of NaCl used ranged anywhere between 1 06 and 600 urn. 
The effect of different lyophilization techniques on pore formation is presented in Table 2. 
Average pore size produced at particular concentrations of NaCl is depicted in Table 3. 



TABLE 1 



Salt content 
(salt-polymer ratio) 


None 


2:1 


9:1 1 


15:1 


25:1 


Interconnecting Pores 


No 


No 


Yes 


Yes 


Yes 


Septum Thickness 


None 


None 


Thick 


Thin 


Very 
Thin 


Void Volume 


None 


<5% 


60-75% 


65-85% 


75-90% 



) 



IABLE_2 



Lyophilization 


No lybph. 


Freeze/lyoph. 


Air Dry 


Lyophilize w€l 


Technique 











wo 97/45532 



-11 - 



PCTAJS97/09067 



Status of Pores 


No 


No 


No 


Interconnecting 




interconnecting 


interconnecting 


interconnecting 


pores 




pores 


pores 


pores 





TABLE 3 





Salt size (nm) 


None 


100-600 


200-400 


400-600 


5 


Pore size i\xm) 


<20 


50-750 


150-500 


350-700 



The paste was then formulated into scaffolds utilizing either of the two following 
10 techniques. 

(1). The paste was packed into rubber molds which were submerged in a 1 liter bath of 
distilled water for 48 hours at 20**C and stirred gently. The water was changed frequently 
(preferably everj' 8 hours). The water serves both as a nonsolvent which is capable of absorbing 
organic solvent and as a pore former which dissolves the NaCl, leaving pores in the scaffold. 
15 The resulting wet scaffold was lyophilized at room temperature for 24 hours, without drying, in 
order to obtain maximum pore interconnectivity with characteristic ultrathin septa between pores. 
It was observed that freezing or air drying greatly diminishes the ability of the scaffold to exhibit 
optimal pore characteristics. The dry scaffolds were then trimmed and cut to desired shape and 
size. 

20 (2). The paste was packed into rubber molds which were submerged in 1 liter of 1 00 % 

ethanol at 20T, a nonsolvent, for 24 hours with frequent change of ethanol. Ethanol only 
absorbs the organic solvent while leaving the salt crystals intact. The scaffold was then cut or 
pressed into the desired shape or size (alternatively the scaffold was cut and shaped after the 
incubation with water). Pore formation was achieved next by submerging the scaffold in 1 liter 

25 distilled water for 24 hours. The water was changed every 8 hours. It was observed that the 
scaffold begins to float during the final stages of salt leaching. The resulting wet scaffold was 

lyophilized to dryness. 

The final scaffold was then sterilized either by standard^thylene oxide gas sterilization or 
steam autoclave at 250T for 30 minutes with a 15 minute dry cycle, after which it was used for 
30 in vitro cell seeding or in vivo implantation. 

h) HYAFF-1 Id75 Scaffold Fabrication Techniow: 



wo 97/45532 PCT/US97/09067 

-12- 

Polymer scaffolds are prepared from HYAFF-1 lp75, a 75% esterified form of hyaluronic 
acid using the following technique. 

A known amount of HYAFF-1 lp75 is dissolved (12 hours, room temp.) in an organic 
solvent (preferably dimethyl sulfoxide, DMSO) to make a 10% (w/v) solution. Presieved NaCl 
crystals of desired size or size ranges (narrow or wide ranges, anywhere between 1 06-600 urn) 
are mixed (5 min., room temp.) With the polymer solution at salt to polymer dry wejght ratio of 
9: 1 or 1 5 : 1 (w/w) to create a slurry or a paste-like mixture. The paste is packed into rubber 
molds and submerged in 100% ethanol for 48 hours with frequent change of EtOl I. Pore 
formation is achieved next by submerging the scaffolds in distilled water (1 L) with stirring for 
30 minutes with frequent change of water. Water treatment is stopped when the foams have 
floated and expanded slightly. HYAFF-1 lp75 loses integrity and structure if left in water for 
extended periods of time, but 30 minutes has been found to be enough time for complete salt 
leaching while maintaining scaffold structure and integrity. The slight expansion causes NaCl to 
be leached out much faster than HYAFF-1 1 scaffolds. The resulting wet scaffold is lyophilized 
and trinuned. 

2. Treated Scaffold preparation: 

In some experiments, the scaffold was dipped, injected, chemically immobilized, or 
sprayed with drugs, peptides, proteins, cytokines, growth factors, oligonucleotides, antisense 
oligonucleotides, DNA, or polymers prior to in vitro or in vivo application. This process 
formulated a hybrid structure that, depending on the molecule or polymer used, targeted cell 
migration, adhesion, commitment, proliferation, or differentiation. The following non-limiting 
examples of treated scaffolds were prepared. 

a) Bone morphogenic protein scaffolds: Scaffolds were produced as described above 
except that lO^g of recombinant human bone morphogenic protein (rhBMP) was adsorbed on to 
the scaffold by dip coating the scaffold with a 0.1 - 1000 Mg/ml BMP solution and air drying for 
30 minutes in a laminar flow hood. Alternatively, 0. 1 - 1 000 \iglm\ BMP in collagen type I 
suspension at 4"C was added to the scaffold and then gelled at 37''C for 1 hour. 

b) Dipped protein scaffolds: The preformed scaffold was dipped in a solution containing 
a drug. A small volume of fluid (e.g. solvent, such as water, DMSO. etc.) containing a known 
amount of drug was coated or sprayed onto the scaffold. The solvem was evaporated by air- 
drying, application of vacuum, lyophilization,€tc. 



wo 97/45532 PCT/US97/09067 

-13- 

c) Chemical linking of drug to scaflfold: A drug was chemically linked to HYAFF 
scaffold. HYAFF surface is activated with coupling agents which bind to OH, NH2» SH or 
COOH groups on the HYAFF. The coupling agent which was bifunctional was reacted with OH, 
NH2, SH or COOH groups on a drug to achieve binding. In one example, CDI (a 
heterobifunctional coupling agent) in acetone or EDC (a heterobifunctional coupling agent 
similar to CDI except that it is soluble in water) in water was used to attach to OH groups on the 
HYAFF. The other end of the EDC or CDI reacted with NH. or COOH groups on the drug or 
peptide to achieve covalent linkage. 

d) A drug that was soluble in DMSO, NMP, etc. (but insoluble in the non-solvent water, 
ethanol, etc.) was mixed with the HYAFF/DMSO or NMP, etc. solution so as to achieve 
incorporation of drug or peptide in the bulk of the HYAFF material. A carrier molecule or 
excipient (albumin, dextran, carboxymethyl cellulose, etc.) was also incorporated. 

e) A drug was added to a preformed HYAFF scaffold by the use of a gel-forming 
material. For example growth factors, BMPs, etc. were added to liquid collagen gels maintained 
at temperature. The growth factor/liquid gel was coated, dipped or sprayed onto the HYAFF 
scaffold. The scaffold/gel construct was then warmed to room temp. (20'*C) or higher (body 
temp. 37°C) for between 20 minutes and two hours in order to effect gelling and entrapment of 
the drug or peptide. 

J) In some experiments, HYAFF scaffold was also sprayed, dipped or coated with a 
second polymer including HYAFF, PLLA, PGA, etc., that contained a drug or peptide. This 
enabled release of the drug and tailoring of scaffold degradation rate. 

g) In some experiments, the HYAFF scaffold was also made with degradable 
microspheres comprised of PLLA, PGA, PLGA, etc., which slowly degraded to form pores. As 
the pores opened, tissue ingrowth occurred. This approach allowed staged tissue invasion in 
cases where early ingrowth was to be discouraged (e.g. infection, -etc.). 

FiTftm ple 1: In vitro ceil growth- pr nUfcration and differentiation on polvmer scaffolds. 

Polymer scaffolds were fabricated as described in Example 1, using sieved NaCl -crystals 
(212 - 600 urn) and both 9:1 and 15:1 salt to polymer dry weight ratios. The scaffolds were 
trimmed to produce cylinders having a 5 mm diameter by 3 mm thickness and autoclaved for 
sterility. The scaffolds were then prewet in 70 % ethanol, and rinsed in sterile PBS. Primary rat 
calvarial osteoblasts isolated from 7 day pups by sequential enzymatic digestion in 1 .37 



wo 97/45532 PCT/US97/09067 

-14- 

mg/ml/collagenase/0.25% trypsin were seeded onto scaffolds at a density of 100,000 cells / 20 uj 
media / scaffold. The cells were allowed to penetrate the pores for 30 minutes, prior to the 
addition of Ham's F12 + 1 0 % FBS (Gibco or Sigma) alone or supplemented with 50 ug/ml 
ascorbic acid (Sigma) and 10 mM p-glycerophosphate (Sigma). The cells were maintained in the 
scaffold apparatus under tissue culture conditions for up to 11 weeks. Cell viability and 
attachment were assessed by fluorescent microscopy using a Live / Dead Eukolight viability kit 
(Molecular Probes, Inc., Eugene, OR). Under scanning electron microscopy (SEM) and light 
microscopic evaluation, the polymer scaffolds showed interconnecting pores 200 to 600 microns 
in diameter. The 15:1 polymer scaffolds showed greater pore interconnectivity and a thinner 
septum between pores than the 9:1 scaffolds. Pilot studies showed that autoclaving did not alter 
scaffold geometry or induce degradation. Osteoblasts seeded onto polymer scaffolds remained 
viable at 1 , 6, and 11 weeks as evidenced by greater than 95 % fluorescence staining for live 
versus dead cells. Increased fluorescence intensity at later lime points also suggested significant 
cell proliferation, although this was not quantified. Scaffolds seeded with osteoblasts cultured in 
ascorbic acid / phosphate supplemented media showed nodules of calcification at 4 weeks and 
beyond, suggesting that HYAFF scaffolds support differentiation. 

Fnamplc 3: J« vivo cell m iprafinti and growth on imnlanted scafffolds. 
In vivo results demonstrated that HYAFF scaffolds can support bone formation. In one 
study, scaffolds (4 mm diameter, 1 mm thickness) were implanted into 4 mm full thickness rat 
cranial defects and harvested after 3 weeks. Empty rat cranial defect sites served as negative 
controls. Upon macroscopic examination after sacrifice, the control empty defect site remained 
largely devoid of tissue. Histological analysis revealed a thin fibrous connective tissue sheath 
adjacent to the dural surface and continuous with the original cranial bone. For HYAFF scaffold 
sites, the implant felt semi-rigid upon examination and was intimately connected with the 
surrounding cranial bone. Histologically, the entire implant was filled with invading tissue. The 
polymer scaffold showed minimal signs of degradation. The scaffold pores were completely 
invaded and contained numerous tightly-packed fibroblastic cells with occasional blood vessels. 
The newly formed tissue in the pores stained positive for collagen and mineral, as evidenced by 
fast green and von Kossa staining. In some larger pores islands of bone trabecule were 
observed. Trabeculae consisted of a row of osteoblast-like cells over an osteoid seam adjacent to 
darkly stained mineral matrix. These results provide concrete evidence that HYAFF scaffolds 



wo 97/45532 PCT/US97/09067 

-15- 

alone are osteoconductivc (ability to support bone ingrowth), in bony defects. 

Example 4: Peptide treated scaffolds capable of 
inducing and sustaining cellular differentiation. 

5 

Addition of exogenous factors to the scaffold, such as drugs, peptides, or proteins, can 
additionally enhance target tissue formation, especially for large, non-healing, critical-sized 
defects. Examples of such drugs, peptides, or proteins are provided in Example 1 above. 

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor 
10 beta (TGFp) superfamily proteins involved in the induction of cartilage and bone. These 

osteogenic and chondrogenic proteins are capable of committing undifferentiated mesenchymal 
stem cells into bone and cartilage forming cells. Scaffolds adsorbed with 10 ug bone 
morphogenetic protein (rhBMP-2) were implanted into rat 8 mm diameter critical-sized cranial 
defects to assess bone formation at a bony site. In addition, similar scaffolds were implanted into 
15 subcutaneous tissue to assess ectopic bone formation. After 3 wrecks, scaffolds at both sites 
displayed significant bone formation in pores that were filled with mineralizing tissue, as 
evidenced histologically with von Kossa staining. BMP-2 scaffolds exhibited more tissue 
ingrowth and mineralization than untreated scaffolds. 

Example S: Preparatio n of a two-phase scaffold, 

20 

A two-phase scaffold was prepared by adding a hydrogel to the solution instead of adding 
a soluble crystal, protein, or microsphere to the HYAFF solution. For example, HYAFF was 
dissolved in DMSO and preformed microspheres were added. Agarose microspheres were 
formed through a hot-melt techniques and sieved to a size of 100-700 microns. The HYAFF 

25 solution and microsphere were mixed and molded into a prescribed shape. The resulting mold 
was then immersed in a bath of water or ethanol which did not dissolve the microspheres but did 
extract the solvent. Similarly, the solvent was extracted by drying under ambient conditions, 
with slightly elevated temperature (<70C) and/or with a gentle vacuum. After or during solvent 
evaporation/air drying the construct was lyophilized to affect f\ill solvent removal and scaffold 

30 formation. The resulting scaffold contained entrapped microspheres which constituted 40-90% 
of the scaffold volume. 70-90% of the void volume contained microspheres with the remainder 
being empty due to microsphere loss. Following lyophilization, the microspheres were 
dehydrated and shrank to 5-20% of their original size. The microspheres were rehydrated by 
immersion in water or placement in tissue. The rehydration resulted in microspheres which were 



PCT/US97/09067 

WO 97/45532 

-16- 

90-95% of their original starting size (e.g. after initial fabrication). Microspheres were fabricated 
from a range of hydrogel materials including agarose, alginate, chitosan, collagens type I, IV, 
etc Matrigel,laminin.etc. The hydrogel microspheres were also covalemly linked with drugs 
and peptides using the coupling strategy described above. Likewise, drugs or peptides were 
™xed with the hydrogel during or after microsphere formation. The advantage of a two-phase 
scaffold is that a range of microspheres containing a range of drugs or peptides (including several 
difTerem drugs and peptides together) can be incorporated in the scaffold for various purposes 
(e.g. antibiotics to treat infection, growth factors to stimulate tissue growth, BMPs to stimulate 
bone or cartilage induction). 

I 

jf^^r-rlr^^: rnmparis>ri ^ft^ntinn of BMP in Wvahir^niy (HA) 

, ... f.„Pn,rira.ior, : Poly-L-lactic acid (PLLA, MW 100kD;Polysciences,lnc.) and 
5 derivatized hyaluronic acid (HA. Fida Advanced Biopolymers, Italy) scaffolds were prepared 
using solvent casting/particulate leaching and phase inversion/particulate leaching techniques, 
respectively. A 20% solution of PLLA in methylene chloride and a 10% solution of HA in N- 
methyl pyrrolidone were each mixed with sodium chloride crystals (106-600 um) at a salt to 
polymer ratio (w/w) of 15:1 . The PLLA mixture was molded, air dried, washed in distilled 
,0 water and dried, all for 24 hours. The HA mixture was washed in distilled water and 

lyophilized, each for 48 hours. Scaffolds were trimmed to a thickness of 1 .5 mm and a diameter 
of 5 mm, and sterilized by steam autoclaving. 

. p^^^..c,,ff-,frfr..e,.„.r/>....r.n-o«: Recombinant human bone morphogenetic protein-2 
25 (rhBMP2, a gift from Genetics Institute, 4.4,g) was coated on HA and PLLA scaffolds in 3 
ways- 1) BMP precoating and drying (HA P or PLA P), 2) BMP and collagen gelled together m 
situ (HA SCB or PLA SCB), and 3) BMP precoating followed by the addition of collagen (PC). 
For all three preparations, scaffolds were prewet in ethanol and rinsed in sterile saline and loaded 
with 4.4 ug rhBMP2. For collagen containing scaffolds, 25 ul collagen 1 (Vilrogen, Collagen 
30 Corp.) was used. Scaffolds (HA S or PLA S) alone served as a control. 

order to assess the amount and bioactivity of rhBMP2 released from scaffolds, an alkahne 



wo 97/45532 PCT/US97/09067 

.17" 

phosphatase assay was used. C3H10T1/2 murine embryonic fibroblasts (ATCC) were cultured , 
in Basal Eagle media (Sigma) with 10% FBS. Scaffolds were placed on membrane inserts (3.0 
um pore size, Fisher) and incubated in 24 well plates containing 12,500 cells/cm'. Scaffolds 
were incubated with cells for either 24 or 48 hours and transferred to freshly plated cells after 

5 each time point for up to 48 days. Cells that had been incubated with scaffolds containing BMP 
were cultured for a total of 4 days. At each time point, cells were lysed in 0.1% Triton X-100 
buffer and assayed for alkaline phosphatase activity using p-nitrophenol phosphate and read 
spectrophotometrically at 410 nm. Specific activity was normalized by total protein and 
expressed as ug/hr/mg protein. Scaffold alone (S), cells alone (control), and collagen gel alone 

10 (collagen) served as negative controls, while one time dose of soluble 1 ug/ml BMP served as 
positive control. 

4. Results: The release of rhBMP-2 by the various scaffolds was determined by the ability of the 
scaffold to stimulate stem cell induction. Hyaluronic acid scaffolds released minimal levels of 
rhBMP-2 as assessed by their inability to stimulate stem cell induction (Fig. 1), Even after 14 
days in vitro, little induction was seen. In contrast, PLLA scaffolds and collagen gels released 
significant levels of BMP for up to 2 weeks (Fig. 2). This level of induction was comparable to 
that seen with 1 ug soluble BMP. These results demonstrate that scaffolds can be engineered to 
locally sequester BMP and suggest that hyaluronic acid scaffolds are superior to poly-L-lactic 
acid or collagen in their ability to retain BMP. 

Scaffolds that sequester BMP at the repair site may show superior bone healing or fusion. 
Increased BMP concentrations within the scaffold should promote more vigorous cell invasion 
and bone induction. BMP is available within the scaffold to act locally due to decreased 
diffusion of BMP out of the scaffold and into the surrounding tissues or bloodstream. Loss of 
BMP due to diffusion may not only decrease the scaffold's potency, but also lead to potential 
bone formation at other unwanted sites. Enhanced BMP retention in HA may be attributed in 
part to ionic interactions where the negatively charged side groups in HA interact with the 
positively charged N-terminal region of rhBMP2. These results demonstrate that scaffold 
chemistry is important in sequestering BMP and that hyaluronic acid scaffolds are superior to 
poly-L-laetic acid or collagen in their ability to retain BMP. 

Each of the foregoing patents, patent applications and references is herein incorporated by 



20 



25 



wo 97/45532 PCT/US97/09067 

-18- 

reference in its entirety. Having described the presently prefened embodiments, in accordance _ 
with the present invention, it is beHeved that other modifications, variations and changes will be 
suggested to those skilled in the art in view of the teachings set forth herein. It is, therefore, to be 
understood that all such variations, modifications, and changes are believed to fall within the 
5 scope of the present invention as defined by the appended claims. 



We claim: 



wo 97/45532 



- 19- 



PCT/US97/09067 



Claims 



1 . A method for forming a substrate for cell growth comprising: 

5 dissolving a water-insoluble hyaluronic acid derivative in a first solvent, 

forming a mixture of the first solvent, the water-insoluble hyaluronic acid 
derivative and a pore forming agent that is insoluble in the first solvent, and 

contacting the mixture with a second solvent, wherein the water-insoluble 
hyaluronic acid derivative is insoluble in the second solvent but the pore forming agent is soluble 
10 in the second solvent, whereby the first solvent and the pore forming agent are extracted from the 
mixture to form a porous scaffold of the water-insoluble hyaluronic acid derivative. 

2. The method of claim 1 , wherein the pore forming agent is sized so as to leave 
voids sufficient to permit cell ingrowth into the scaffold when the pore forming agent is extracted 

15 from the mixture. 

3. The method of claim I further comprising dr>'ing the scaffold under vacuum at a 
temperature between 4°C and 3 7T. 

20 4. The method of claim 3 wherein the scaffold is dried at ambient temperatures. 

5. The method of claim 1 wherein the water-insoluble hyaluronic acid derivative is a 
covalent conjugate of hyaluronic acid^sterified with a benzyl moiety. 

25 6. The method of claim 1 fiarther comprising coating the scaffold with a bioerodable 

polymer. 

7. A substrate for cell growth comprising: 

a scaffold of water-insoluble derivatized hyaluronic acid defining interconnected 
30 pores of sufficient size to permit mammalian cell ingrowth into the pores, wherein the 

derivatized hyaluronic acid is a covalent conjugate of hyaluronic acid and a water-insoluble 
moiety that renders the conjugate insoluble in water. 



wo 97/45532 PCT/US97/09067 

-20- 

8. The substrate of claim 7 wherein the covalent conjugate comprises an ester bond, 
via a carboxy group of the hyaluronic acid, 

9. The substrate of claim 7 wherein the covalent conjugate comprises an amide bond 
5 via a carboxy group of the hyaluronic acid. 

10. The substrate of claim 7 wherein the covalent conjugate is hyaluronic acid 
esterified with a benzyl moiety. 

10 11. The substrate of claim 1 0 wherein the scaffold is coated with a bioerodible 

polymer. 

12. The substrate of claim 7 further comprising mammalian cells within at least some 
pores of the scaffold. 

15 

1 3 . The substrate of claim 7, wherein the scaffold is a two-phase scaffold 
incorporating hydrogel. 

14. The methods of claim 1 further adding a compound selected from the group 
20 consisting of drugs, growth factors, peptides, proteins, cytokines, oligonucleotides, antisensc 

oligonucleotides, DNA and polymers. 

15. The method of claim 1 4, wherein the compound is added by coating the porous 
scaffold with the compounds. 

25 

1 6. The method of claim 14, wherein the compound is added by covalent attachment 
to the porous scaffold. 



wo 97/45532 



PCTAJS97/09067 



Fig. 1. 





160 - 




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140 - 




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120 - 


c 




100 - 




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80 - 


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40 - 








20 - 




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12 16 20 24 28 32 36 40 44 48 



time (days) 



Fig. 2. 





160 - 




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100 - 


a 




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80 - 


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60 - 






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PLAP 
PLA PC 




I ' I ' I ■ I ■ I ■ I ■ 1 ■ I ■ I ■ I ' I 

8 12 16 20 24 28 32 36 40 44 46 



time (days) 



1/1 



INTERNATIONAL SEARCH REPORT 



tnte. .ional Application No 

PCT/US 97/09067 



CLASSIFICATION OF SUBJECT MATTER 

PC 6 C12N5/00 A61L27/0O 



Aocording to International Patent ClaMtfication (IPC) or to both national ctawifioatton and IPC 



FIELDS SEARCHED 



Minimum documentation Marched (claaaitication »y»!em foltowod by dasaifioation aymbote) 

PC 6 C12N A61L 



DocumenUUon •earchwi othar than minimum dooumanlation to the eirtent that.uch documentt are included in the fields tearohed 



Eleotronic data base oonauKed during the international search (name of data bate and, where practical. »eareh ter me ueed) 



C. DOCUMENTS CONSIDERED TO BE RELEVANT 



Category • Citation of document, with indication, where appropriate, of the relevant paeiaget 



Releveflt to claim No. 



WO 96 10426 A (YAMANOUCHI PHARMACEUTICAL 
CO. LTD.) 11 April 1996 
-& EP 0 784 985 A (YAMANOUCHI 
PHARMACEUTICAL CO. LTD.) 23 July 1997 
see page 4, column 5, line 13 - column 6, 
line 30; claims 

see page 7, column 12, line 46 - line 51 

WO 94 17840 A (FIDIA ADVANCED BIOPOLYMERS 
S.R.L.) 18 August 1994 
see claims; examples 1-4 

WO 93 11803 A (M.U.R,S.T.) 24 June 1993 
see claims; examples 12,14 



7-9.11 
7-9,11 



1-16 



1-16 



□ 



Further documenta are beted in the continuation of box C. 



m 



Patent family members are litted in annax. 



* Speoial oategoriee of cited dooumenta : 

■A' document defining the general atale of the art which m not 

oonaidered to be of paftioular ivlevanoe 
'E* earlier docurrwnt but publiahed on or aftar the htemational 

fding dale 

V document which may throw doubti on priorily ctalmfe) or 
whioh i» cted to eetablieh the publication date of another 
citalion or other ipeoial reaaon <ai specified} 

'O* document refenvtg to an oral dwolosufie, use, exhibitton or 
other means 

'P* document pubfshed prior to the international filing date but 
later than the priority date claimed 



"T" later dooumcnl pUbliBhed after the international liKng date 
or priority date and not in confliel with the appfeoatbn but 
cited to undervtand the principle or theory underlying the 



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

*Y* document of particular relevanoe; the claimed invention 

oannot be considered to involve an inventive step when the 
document is oombined wih one or more other such docu- 
ments, suoh oombination being cbmus to a peraon sKMIed 
in the art. 

*&' dooumamt member of the same patent family 



Date of the aolual completion of theintemational eearoh 



6 October 1997 



Date of mailing of the international search report 



2 9. 10. 97 



Name and mailing address of ttte ISA 

European Patent Office, P.B. SB18 Patentlaan 2 
NL - 2280 HV RijawiiK 
Tel. (♦3170) 340*2040, Tx. 31 651 epo nl. 
Fax: (+31-70)340-3016 



Authorized offioer 



Ryckebosch. A 



Fonn PCT/lSA/310 (second thost) (July 19S2) 



INTERNATIONAL SEARCH REPORT 

tnformaUon on patenl family membets 



Inte «onat Application No 

PCT/US 97/09067 



PsAenX document 
cited in search report 



Publicalion 
date 



Patent family 
member^B) 



Publication 
date 



wo 961G426 A 



WO 9417840 A 



11-04-95 



AU 3577895 A 
CA 2200052 A 
EP 0784985 A 



18-08-94 



IT 1263144 B 

AU 6001494 A 

EP 0682534 A 

JP 8506497 T 



26-04-96 
11-04-96 
23-07-97 



01-08-96 
29-08-94 
22-11-95 
16-07-96 



WO 9311803 A 



24-06-93 



IT 


1254704 B 


G9-10-95 


AU 


669147 B 


30-05-96 


AU 


3346693 A 


19-07-93 


BG 


98863 A 


31-05-95 


EP 


0618817 A 


12-10-94 


FI 


942894 A 


18-08-94 


HU 


68680 A 


28-07-95 


JP 


7502430 T 


16-03-95 


NO 


942330 A 


17-08-94 


NZ 


246575 A 


24-04-97 


US 


5520916 A 


28-05-96 



Foim PCTrtSA^IO (paten* »«T>rty aniwK) <July 1992) 



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