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WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




PCX 

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) International Patent Classification ^ : 
A61L 27/38 



Al 



(11) International Publication Number: WO 00/37 124 

(43) International Publication Date: 29 June 2000 (29.06.00) 



(21) International Application Number: PCT/1B99/02077 

(22) International Filing Date: 21 December 1999 (21.12.99) 



(30) Priority Data: 

PD98A000298 



2 1 December 1 998 (2 1 . 1 2.98) IT 



(71) Applicant (for all designated States except US): FIDIA AD- 

VANCED BIOPOLYMERS, S.R.L. [IT/IT]; Via de Carpen- 
tieri, 3. 1-72100 Brindisi (IT). 

(72) Inventors; and 

(75) Inventors/Applicants (for US only): RADICE. Marco [IT/IT]; 
Via Mantova, 20, 1-35143 Padova (IT). PASTORELLO, 
Andrea [IT/IT]; Via Alessandro Volta. 3/B, 1-35031 Abano 
Terme (IT). PAVESIO, Alcssandra [IT/IT]; Via Decorati al 
Valorc Civile. 159, 1-35100 Padova (IT). CALLEGARO, 
Lanfranco [IT/IT]; Via Monte Grappa, 6. 1-36016 Thiene 
(IT). 

(74) Agents: CRUMP, Julian, Richard, John et al.; fJ Cleveland, 
40-43 Chanceiy Lane, London WC2A IJQ (GB). 



(81) Designated States: AE. AL, AM. AT. AU, A2, BA, BB, BG. 
BR, BY. CA. CH. CN, CR. CU, CZ, DE. DK, DM, EE, 
ES, n, GB, GD, GE, GH, GM, HR, HU. ID, IL. IN, IS, JP, 
KE, KG. KP, KR, KZ. LC, LK, LR, LS. LT. LU. LV. MD, 
MG. MK. MN. MW, MX, NO. NZ. PL, PT, RO, RU, SD, 
SE, SG. SI, SK. SL. TJ. TM. TR, TT. TZ. UA. UG. US. 
UZ, VN. YU. ZA. ZW, ARIPO patent (GH. GM. KE, LS, 
MW. SD, SL. SZ, TZ, UG. ZW). Eurasian patent (AM. AZ, 
BY. KG. KZ. MD. RU. TJ. TM), European patent (AT, BE, 
CH, CY. DE. DK. ES, FI. FR. GB. GR, IE, IT, LU, MC, 
NL, PT, SE), OAPl patent (BF, BJ. CF, CG, Q. CM, GA. 
GN, GW, ML, MR. NE, SN, TD, TG). 



Published 

With international search report. 

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



(54) Titie: INJECTABLE HYALURONIC ACID DERIVATIVE WITH PHARMACEUTICALS/CELLS 



(57) Abstract 

Disclosed is an injectable, biocompatible and biodegradable composition, comprising at least one hyaluronic acid benzyl ester or 
auto-cross-linked derivative, in combination with at least one mammalian cell and/or at least one pharmacologically or biologically active 
substance and/or micro-particles such as fibres, granules, microspheres or sponge fragments of a hyaluronic acid derivative. 



FOR THE PURPOSES OF INFORM A TION ONLY 
Codes used lo identify States parry to the PCT on the front pages of pamphlets publishing international applications under the PCT. 



AL 


Albania 


ES 


Spain 


ts 


Lesotho 


SI 


Sbvenia 


AM 


Annenia 


n 


Fmland 


LT 


Lithuania 


SK 


Slovakia 


AT 


Austria 


PR 


France 


LU 


Luxembourg 


SN 


Senegal 


AU 


Australia 


OA 


Gabon 


LV 


Latvia 


sz 


Swaziland 


AZ 


Azeftaijan 


GB 


United Kingdom 


MC 


Monaco 


TD 


Chad 


BA 


Bosnia and Herzegovina 


G£ 


Georgia 


MD 


Republic of Moldova 


TG 


Togo 


BB 


Barbados 


GH 


Ghana 


MG 


Madagascar 


TJ 


Tajikistan 


BE 


Belgium 


GN 


Guinea 


MK 


The forrocr Yugoslav 


TM 


Turkmenistan 


BF 


Burlcina Faso 


GR 


Greece 




Republic of Macedonia 


TR 


•niikey 


BG 


Bulgaria 


HU 


Hungary 


Ml 


Mali 


TT 


Trinidad and Tobago 


BJ 


Benin 


IE 


Ireland 


MN 


Mongolia 


UA 


Ukraine 


BR 


Brazil 


IL 


Israel 


MR 


Mauritania 


UG 


Uganda 


BY 


Belarus 


IS 


Iceland 


MW 


Malawi 


US 


United Suites of America 


CA 


Canada 


IT 


Italy 


MX 


Mexico 


uz 


Uzbekistan 


CF 


Central African Republic 


JP 




NE 


Niger 


VN 


Vict Nam 


CG 


Congo 


K£ 


Kenya 


NL 


Netherlands 


YU 


Yugoslavia 


CH 


Switzerland 


KG 


Kyrgyzstan 


NO 


Norway 


ZW 


Zimbabwe 


CI 


Cfltc d*lvoire 


KP 


Democratic People^s 


WZ 


New Zealand 






CM 


Cameroon 




Republic of Korea 


PL 


Poland 






CN 


China 


KR 


Republic of Korea 


PT 


PoiTugal 






CU 


Cuba 


KZ 


Kazakstan 


RO 


Romania 






CZ 


Czech Republic 


LC 


Saint LAicia 


RU 


Russian Federation 






D£ 


Germany 


U 


Liechtenstein 


SD 


Sudan 






DK 


Denmark 


LK 


Sri Lanka 


S£ 


Sweden 






EE 


Estonia 


LR 


Liberia 


SG 


Singapore 







wo 00/37124 



PCT/1B99/02077 



INJECTABLE HYALURONIC ACID DERIVATIVE WITH PHARMACEUTICALS/CELLS 



SUBJECT OF THE INVENTION 

The present invention is directed to an injectable, biocompatible and biodegradable 
composition, comprising at least one hyaluronic acid benzyl ester or auto-cross-linked 
derivative, in combination with at least one mammalian cell and/or at least one 
10 pharmacologically or biologically active substance and/or micro-particles such as fibres, 
granules, microspheres or sponge fragments of a hyaluronic acid derivative. 

BACKGROUND OF THE INVENTION 

Although injectable compositions and carriers for such compositions have been known in 
the art, there still exists a need for injectable compositions which are biocompatible, are 
1 5 biodegradable, offer protective aspects to the active component, and provide enhanced 

bioavailability of the active components. This is important, for instance, in the field of joint 
cartilage repair. 

The aim of joint cartilage repair is to restore the surface of the joint, reduce pain and 
prevent fiirther deterioration of the tissues. Many methods have been applied to date for 
20 the treatment of cartilage defects, each of which has presented disadvantages (Tom Minas 
et al,, "Current Concepts in the treatment of Articular Cartilage Defects", Orthopedics, 
June 1997, vol. 20, No. 6). 

The marrow stimulation technique consists of reaching subchondral bone tissue ar«as by 
means of abrasion or perforation, thus stimulating the formation of a fibrin clot containing 
25 pluripotent stem cells. The clot subsequently differentiates and takes shape, forming 

fibrocartilage repair tissue However, this tissue does not have the mechanical properties or 
the physiological and structural characteristics of healthy, lasting joint cartilage. 

Another technique consists of implanting into the site of thexiefect a piece of periosteal 
and perichondral tissue taken, for example, from the rib cartilage. Such tr^tment does 
30 trigger the development of hyaline cartilage, but the repair tissue is poorly integrated with 
the surrounding healthy tissues and the implanted tissue subsequently becomes ossified. 



wo 00/37124 PCT/IB99/02077 

"2- 

Autologous and homologous osteochondral grafts are invasive, require complex surgical 
techniques and carry the risk of, for example, viral infection. 

Other attempts to reconstruct the joint cartilage consist of implanting synthetic matrices 
with allogenic chondrocytes dispersed within them, or growth factors able to stimulate the 
5 proliferation of the chondrocytes. These methods require that the cartilage tissue is grown 
in vitro and then implanted into the defect. The synthetic matrices most commonly used 
are collagen gels, matrices of polyanhydrides, polyorthoesters, polyglycolic acid and its 
copolymers. The chief disadvantage of the use of such matrices is represented by the 
immune response directed against the implanted material. Chondrocytes are known to be 

10 cultured in gel constituted by agarose, hyaluronic acid, fibrin glue, collagen and alginate. 
However, these cultures in gel do not provide the mechanical stability necessary for them 
to adhere to the site once implanted and to allow the reconstruction of the cartilage 
structure. Moreover, chondrocyte cultures in substances such as fibrin de-differentiate into 
cells which appear to be similar to fibroblasts. Lastly, ahhough gels constituted by 

15 substances such as agarose induce chondrocyte re-differentiation, the use of this compound 
has not been approved for internal applications to humans. 

Joint cartilage defects have also been treated with suspensions of isolated chondrocytes in 
the absence of supporting matrices. It is thought, however, that chondrocytes lose their 
viability and/or do not remain at the site of the defect and that they form fibrocartilage or 
20 islets of cartilage immersed in fibrous tissue (see US Patent 5,723,33 1). 

These disadvantages of the prior art are overcome by the present invention bv providing an 
injectable composition such as one containing chondrocytes or bone marrow stroma cells 
dispersed in a gel containing at least one hyaluronic acid benzyl ester derivative or auto- 
crossiinked derivative. 

25 Various pieces of evidence have emerged in the literature (see enclosed abstract) recently 
concerning the use of cell suspensions for injection purposes, in particular keratinocytes 
for the treatment of chronic ulcers and bums. See Silverman et al, Plast. Reconstr. Surg., 
June 1999, 103(7) 1809-18 (combination of fibrinogen and chondrocytes); Atala et al., J. 
Urol., August 1993, 150 (2 Ptd. 2) p. 745-7 (chondrocyte-alginate gel)). Keratinocyte 

30 cultures can be developed according to various methods cited in the literature (in the 

presence or absence of foetal calf serum, with chemically defined culture medium, etc.). 



wo 00/37124 



PCT/1B99/02077 



These cultures are then vehicled in the host bed suspending them in various media, one of 
the most frequently cited of which is fibrin both of autologous and commercial origin. 
There are considerable disadvantages to the use of such methods. Firstly, the cell 
suspension has to be prepared immediately before use, so the cells have to be stored in a 
5 medium with a different composition from the one used for their application, while other 
problems may arise with the fibrin glue used as a vehicle, particularly when this is not 
autologous. 

These problems are overcome by the present invention by dispersing epithelial cells (such 
as keratinocytes) or derivatives of other embryonic origin in a hyaluronic-acid-based 

1 0 medium for various reasons. The preparation is perfectly biocompatible and biologically 
safe and the cell survival rate is higher than in cell suspensions in completely liquid media. 
This last point in particular is important. In cases where the patient or application site is a 
long distance from the site of production for the component, safe transport becomes a 
problem. The product will inevitably be shaken about during transport damaging the cells, 

15 and this problem needs to be solved. However, when the cells are dispersed in a highly 

viscous medium according to the present invention, this problem is overcome because the 
host medium acts as a cushion. Another advantage derives from the possibility of 
spreading the cell suspension efficiently over the surface to be treated, which is a simpler 
way of applying it than the methods currently used, involving sprays based on fibrin glue. 

20 Another application of the present invention concerns the possibility of suspending the 
cells in the medium and then applying them by injection. Other non-limiting applications 
are the administration of fibroblasts (autologous) for aesthetic surgical purposes or as 
fillers for tissue defects, preparations of adipocytes (autologous, heterologous or 
homologous) for soft tissue augmentation for applications such as the reconstruction of 

25 breasts or other soft body parts, injections of urethral cells such as fibroblastoids or 
cartilage cells for the treatment of urinary incontinence. In all these examples, the 
Hyaluronic acid-based material has the double fimction of acting as a vehicle for injections 
and of protecting the cell preparation during transport. 



30 



As is known, hyaluronic acid plays a vital role in many biological processes such as tissue 
hydration, proteoglycan organization, cell differentiation, proliferation and angiogenesis (J. 
Aigner et al. Biomed. Mater. Res. 1998, 42, 172-lSl). Hyaluronic acid derivatives 



wo 00/37124 



PCT/IB99/02077 



maintain all the properties of said glycosaminoglycan, with the advantage of being able to 
be processed in various forms and having solubility and degradation times which vary 
according to the type and percentage of derivation (EP 0216453 Bl). Moreover, the 
hyaluronic acid derivatives offer new properties due to the insertion of specific molecules 
5 in the structure of the hyaluronic acid. For example, the sulfated derivatives of hyaluronic 
acid have anticoagulant properties and are resistant to hyaluronidase (WO 95/25751). It 
has been demonstrated that said compositions do not trigger immune responses by the 
organism and the chondrocytes they contain maintain their phenotype. Hyaluronic acid 
derivatives are not cytotoxic and allow the synthesis of components of the extracellular 

1 0 matrix that are necessary for the development of the cartilage tissue. Moreover, said 

derivatives do not represent a simple vehicle for the cells but are able to stimulate their 
poliferation and, as they degrade, allow the development of the cells into thr-ee-dimensional 
structures. Besides stimulating the growth of implanted cells, the hyaluronic acid 
derivatives are able to create an extracellular environment similar to that of mammal 

1 5 foetuses which stimulates the regeneration of tissues. Moreover, as the hyaluronic acid 

derivatives degrade, they release oligomers, stimulating the recruitment of progenitor cells 
of chondrocytes and favouring their development towards the chondrocyte cell line. 

It is known that hyaluronic acid derivatives can be used as three-dimensional, solid 
scaffolds in the form of non-woven fabrics, sponges, granules, microspheres, tubes and 

20 gauzes to grow stem cells in vitro (WO 97/1 8842), in the form of non-woven fabrics 
associated with a perforated membrane for the growth in vitro of fibroblasts and 
keratinocytes (WO 96/33750) and in the form of non-woven fabrics for the growth of 
chondrocytes (J. Aigner et al., L. Biomed. Mater. Res., 1998, 42, 172-181). However, to 
date, nobody has made an injectable gel containing hyaluronic acid derivatives and 

25 mammalian cells, such as chondrocyte cells, that allows the surgeon to use only mildly 
invasive surgical techniques, such as endoscopic surgery, enabling the cells to be 
incorporated in a composition to survive transport and completely fill irregularly-shaped 
lesion sites. 

Unlike the method of seeding of cells on solid supports, in the present invention the cells 
30 are evenly dispersed in all three dimensions throughout the composition in the form of a 
gel made according to the present invention. Said compositions allow the regenerated 
tissue to integrate perfectly with the cartilage tissue surrounding the defect. The 



wo 00/37124 



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PCT/IB99/02077 



compositions according to the present invention can be used to advantage for the treatment 
of both superficial and deep cartilage defects. Superficial defects are those affecting the 
cartilage tissue alone, while deep defects are those which also involve the subchondral 
bone tissue and the layer of calcified cartilage between the subchondral bone tissue and the 
cartilage. 

DETAILED DESCRIPTION OF THE INVENTION 

The present invention concerns injectable, biocompatible and biodegradable compositions 
containing at least one hyaluronic acid benzyl ester derivative and/or auto*crossIinked 
derivative, at least one pharmacologically and/or biologically active substance, such as a 
growth factor, and/or at least one mammalian cell, particularly chondrogenic cells. 

1. The Hyaluronic Acid Component 

The present invention, therefore, describes injectable biocompatible compositions based on a 
benzyl ester of hyaluronic acid or on an auto-cross-linked derivative of hyaluronic acid, used 
singly or in mixtures with one another, characterized by high biocompatibility. The materials 
are also completely biodegradable and do not need to be removed from the application site, 
thus avoiding a second surgical operation. When prq)ared in the form of gels, the cross-linked 
derivatives present materials with significantly greater viscosity than the unmodified polymer 
and with variable degradation times. 

The term "hyaluronic acid" (also referred to as "HA" hereinafter) is used in literature to 
designate an acidic polysaccharide with various molecular weights constituted by resides 
of D-glucuronic acid and N-acetyl-D-glucosamine, which naturally occur in cellular 
surfaces, in the basic extracellular substances of the connective tissues of vertebrates, in the 
synovial fluid of joints, in the vitreous humor of the eye, in the tissue of the human 
umbilical cord and in cocks* comb. 

Hyaluronic acid plays an important role in the biological organism, firstly as a mechanical 
support of the cells of many tissues, such as the skin, the tendons, the muscles and cartilage 
and it is therefore the main component of the extracellular matrix. But hyaluronic acid also 
perfbrms other functions in the biological processes, such as the hydration of tissues, 
lubrication, cdhilar migration, cell function and differentiation. (See for example, A. Balazs 
et al.. Cosmetics & Toiletries, No. 5/84, pages 8-17). Hyaluronic acid may be -extracted fi-om 
the above-mentioned natural tissues, such as cocks* combs, or also from certain bacteria 



wo 00/37124 PCT/1B99/02077 

-6- 

Today, hyaluronic acid may also be prepared by microbiological methods. The molecular 
weight of whole hyaluronic acid obtained by extraction is in the region of 8-13 millioa 
However, the molecular chain of the polysaccharide can be degraded quite easily under the 
influence of various physical and chemical factors, such as mechanical influences or under the 
5 influence of radiation, hydrolyzing, oxidizing or enzymatic agents. For this reason, often in 
the ordinary purification procedures of original extracts, degraded fractions with a lower 
molecular weight are obtained. (See Balazs et al., cited above). Hyaluronic acid, its 
molecular fractions and the respective salts have been used as medicaments and their use is 
also proposed in cosmetics (see for example, the above-mentioned article by Balazs et al., and 
1 0 the French Patent No. 2478468). 

Akhough the term "hyaluronic acid" is commonly used in an improper sense, meaning, as can 
be seen from above, a whole series of polysaccharides with alternations of residues of D- 
glucuronic acid and N-acetyl-D-glucosamine with varying molecular weights or even 
degraded fractions of the same, and although the plural form "hyaluronic acids" may seem 
1 5 more appropriate, the discussion herein shall continue to use the singular form to refer to 
hyaluronic acid in its various forms including its molecular fractions, and the abbreviation 
"HA" will also often be used to describe this collective term. 

The present invention describes injectable compositions containing hyaluronic acid 
derivatives which work as suitable carriers for biological/pharmacological cells or 

20 molecules. Hyaluronic acid derivatives are certainly more suitable than other 

biomaterials/scafFolds known in the prior art. In comparison with biological-derived 
system, such as, for instance, cadaveric acellular material, HA has the advantage to be 
readily available in unlimited supply and not highly immunogenic, such as allogeneic 
donor tissues. In addition, HA is not at risk for cross-contamination for infective diseases, 

25 especially virus derived (HIV, Hepatitis, etc. ). In comparison with more purified 

biological-derived molecules, such as, for instance collagen, proteoglycans and fibrin, or 
biocompatible synthetic polymers, such as, for instance, PLLA/PGA, PTFE, HA has 
different favourable characteristics. First of all, HA is a polysaccharide which shows less 
immunogenic reactions than common proteic- ot proteic-based compounds. Secondly, HA 

30 is commonly found in all mammals species with no modification of the molecular 

structure, thus, is very well known and tolerated by the human body. Third, HA has many 
biological effects, in developing as well as aduh humans, which make the molecule to be 



wo 00/37124 



PCT/IB99/02077 



fundamental in each reparative/regenerative process. Finally, another favourable point is 
that HA is present in almost all tissues/organs of the human body, being a major 
component of the extracellular matrix. This fact, along with the simple composition of the 
polymer, make HA different from many proteic extracellular matrix molecules, such as, for 
5 instance collagen, that are, very often, tissue/organ specific. This last point is very 
important in designing a general and biocompatible delivery vehicle to be used for 
different compartment of the human body, 

2. The Benzvl Ester Derivatives 

The first preferred material of the invention is based on the benzyl ester of hyaluronic add, 
1 0 particularly the 50-75% esters wherein 50% to 75% of the HA carboxyl groups are esterified 
with a benzyl residue. Those benzyl esters wherein 50-75% of the HA carboxyl groups are 
esterified with a benzyl group are referred to as "partial esters", because only a portion of the 
carboxyl groups are esterified and the remaining carboxyl groups are either free or salified 
with an alkaline or alkaline earth metal, such as sodium, calcium or potassium. 

15 Most preferred for the compositions of the invention are the benzyl esters wherein 50% of the 
HA carboxy groups are esterified. The benzyl esters of hyaluronic acid according to the 
invention may be prepared by methods known per se for the esterification of carboxylic acids, 
for example by treatment of fi^ee hyaluronic acid with the alcohol (benzyl alcohol) in the 
presence of catalyzing substances, such as strong inoiganic acids or ionic exchangers of the 

20 acid type, or with an etherifying agent capable of introducing the desired alcoholic residue in 
the presence of inorganic or organic bases. 

The benzyl hyaluronic esters may, however, be preferably prepare to advantage according to 
a particular method described in EP 0 216 453. This method consists of treating a quaternary 
anunonium salt of hyaluronic acid with an etherifying agent, preferably in an aprotic organic 
25 solvent. 

For the preparation of the benzyl esters it is possible to use hyaluronic acids of any origin, 
such as for example, the acids extracted from the above mentioned natural starting materials, 
for example, fi-om cocks* combs. The preparation of such acids is described in literature; 
preferably, purified hyaluronic acids are used. According to the invention, especially used are 
30 hyaluronic acids comprising molecular fractions of the int^ral acids obtained directly by 
extraction of the organic matoials with molecular weights varying within a wide range, for 



wo 00/37124 PCT/IB99/02077 

-8- 

example, from about 90%-80% (M = 1 1 . 7 - 1 0.4 million) to 0.2% (M = 30,000) of the 
molecular weight of the integral acid having a molecular weight of 13 million, preferably 
between 5% and 0.2%. Such fraaions may be obtained with various procedures described in 
literature, such as by hydrolyzing, oxidizing, enzymatic or physical procedures, such as 
5 mechanical or radiational procedures Primordial extracts are therefore often formed during 
these same purification procedures (for example, see the article by Balazs et al., quoted above 
in "Cosmetics & Toiletries"). The separation and purification of the molecular fractions 
obtained are brought about by known techniques, for example by molecular filtration. 

One fraction of purified HA suitable for use according to the invention is for example that 
1 0 known as "non-inflammatory-NIF-NaHA" sodium hyaluronate described by Balazs in the 
booklet "Healon" - A guide to its use in Ophthalmic Surgery, D. Miller & R. Stegmann, eds. 
John Wiley & Sons, N.Y., 81983 : p 5 

Particularly important as starting materials for the benzyl ester are two purified fractions 
obtainable from hyaluronic acid, for example the ones extracted from cocks' combs, known as 

1 5 "Hyalastine" and "Hyalectin". The fraaion Hyalastine has an average molecular weight of 
about 50,000 to 100,000 while the fraction Hyalectin has an average molecular weight of 
between about 500,000 and 730,000. A combined fi^aion of these two fractions has also 
been isolated and characterized as having an average molecular weight of about 250,000 to 
about 350,000. This combined fraaion may be obtained with a yield of 80% of total 

20 hyaluronic acid available in the particular starting material, while the fraction Hyalectin may 
be obtained with a yield of 30% and the fraction Hyalastine with a yield of 50% of the starting 
HA. The preparation of these fractions is described in EP 0 138 572. 

The following Examples describe the preparation of the benzyl esters of HA. 

Example 1 - Preparation of the 50% benzyl ester of hyaluronic acid /HYj- 50% of 
25 esterified carboxvlic groups - 50% of salified carboxvlic -groups fNa^ 

12.4 g of HY tetrabutylammonium sah with a molecular weight of 170,000 corresponding 
to 20 m.Eq. of a monomeric unit are solubilized in 620 ml of dimethysulfoxide at 25°C, 10 
m.Eq.) of benzyl bromide are added and the resulting solution is kept at a temperature of 
30^ for 12 hours. 



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PCT/IB99/02077 



A solution containing 62 ml of water and 9 g of sodium chloride is added and the resulting 
mixture is slowly poured into 3,500 ml of acetone under constant agitation. A precipitate is 
formed which is filtered and washed three times with 500 ml of acetone/water 5: 1 and 
three times with acetone and finally vacuum dried for eight hours at 30°. 

5 The product is then dissolved in 550 ml of water containing 1% of sodium chloride and the 
solution is slowly poured into 3,000 ml of acetone under constant agitation. A precipitate is 
formed which is fihered and washed twice with 500 ml of acetone/water (5:1) and three 
times with 500 ml of acetone and finally vacuum dried for 24 hours at 30°. 8.6 g of the 
partial benzyl ester compound in the title are obtained. Quantitative determination of the 
10 ester groups is carried out using the method of R.H. Cundiff and P.C. Markunas [Anal. 
Chem. 33, 1028-1030,(1961)]. 

Example 2 - Preparation of the 75% benzyl ester of hyaluronic acid (HY) - 75% of 
esterified carboxvlic groups - 25% of salified carboxvlic groups (Na^ 
12.4 g of HY tetrabutylammonium salt with a molecular weight of 250,000 corresponding 
15 to 20 m.Eq. of monomeric unit are solubilized in 620 ml of dimethyl sufoxide at 25®, 2.5 g 
(15 m.Eq) of benzyl bromide are added and the resulting solution is kept for 12 hours at 
30°. 

A solution containing 62 ml of water and 9 g of sodium chloride is added and the resulting 
mixture is slowly poured into 3,500 ml of acetone under constant agitation. A precipitate is 
20 formed which is fihered and washed three times with 500 ml of acetone/water 5:1 and 
three times with acetone and finally vacuum dried for eight hours at 30° 

The product is then dissolved in 550 ml of water containing 1% of sodium chloride and the 
solution is slowly poured into 3,000 ml of acetone under constant agitation. A precipitate is 
formed which is fihered and washed twice with 500 ml of acetone/water 5:1 and three 
25 times with 500 ml of acetone and finally vacuum dried for 24 hours at 30°. 9 g of the 

partial benzyl ester compound in the title are obtained. Quantitative determination of the 
ester groups is carried out using the method of R.H. Cundiff and P.C. Markunas [Anal. 
Chem. 33, 1028-1030, (1961)]. 



30 



Example 3 - Preparation of the 75% ester of hyaluronic acid (HY) - 75% of esterified 
carboxylic groups - 25% of salified carboxylic groups (Na) 



wo 00/37124 PCT/IB99/02077 

-10- 



12.4 g of HY tetrabutylammonium salt with a molecular weight of 80,000 corresponding to 
20 m.Eq. of a monomeric unit are solubilized in 620 ml of dimethylsufoxide at 25**, 2.5 g 
(15 m.Eq.) of benzyl bromide are added and the resuhing solution is kept for 12 hours at 

30°. 

5 A solution containing 62 ml of water and 9 g of sodium chloride is added and the resulting 
mixture is slowly poured into 3,500 ml of acetone under constant agitation. A precipitate is 
formed which is filtered and washed three times with 500 ml of acetone/water 5: 1 and 
three times with acetone and finally vacuum dried for eight hours at 30° 

The product is then dissolved in 550 ml of water containing 1% of sodium chloride and the 
10 solution is slowly poured into 3,000 ml of acetone under constant agitation. A precipitate is 
formed which is filtered and washed twice with 500 ml of acetone/water 5:1 and three 
times with 500 ml of acetone and finally vacuum dried for 24 hours at 30°. 9 g of the 
partial benzyl ethyl ester compound in the title are obtained. Quantitative determination of 
the ester groups is carried out using the method of R.H. Cundiff and P.C. Markunas [Anal. 
15 Chem. 33, 1028-1030, (1961)]. 

3. The Auto (or Inteman Cross-Linked Hyaluronic Acid Derivatives (ACP 
Derivatives) 

The auto cross-linked hyaluronic acid derivatives used in the materials of the present 
invention are described in HP 0 34 1 745. These cross-linked derivatives are inter and/or 

20 intramolecular esters of hyaluronic acid wherein a part of the carboxy groups are esterified 
with hydroxyl groups of the same molecule and/or of different molecules of hy Juronic acid, 
thus forming lactone or intermolecular ester bonds. These "inner" esters, in which there is no 
intervention by OH groups of other alcohols, can also be defined as "auto-^crosslinked 
hyaluronic acid" (also referred to as "ACP") since the formation of a mono- or polymolecular 

25 cross-link is the consequence of the above-mentioned internal esterification. The adjective 

"cross-linked" refers to the crosswise conneaions between the carboxyls and hydroxyls of the 
hyaluronic acid molecules. 

The auto-CTOSslinked products are particularly partial inner esters wherein the percentage of 
"cross-links" varies preferably between 3 to 1 5% of the numbo- of rarboxy groups in the 
30 hyaluronic acid. In the preparation process, the carboxy groups of the HA molecule are 



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activated by the addition of substances capable of inducing such activation. The unstable 
intermediate products obtained from the activation reaaion separate spontaneously, either 
after the addition of catalysts and/or following a rise in temperature, forming the above 
mentioned inner ester bonds with hydroxyls of the same or other hyaluronic acid molecule. 
5 According to the degree of inner esterification desired, either all or an aliquot part of the 
carboxy functions are activated (the aliquot part being obtained by using an excess of 
activating substances or by suitable dosing methods). 

The carboxy groups to be converted into inner ester groups can be activated starting from 
hyaluronic acid containing free carboxy groups, or, preferably, from HA containing salified 
1 0 carboxy groups, for example, metal salts, preferably alkaline or alkaline earth metals, and 
above all with quaternary ammonium salts, such as those described hereafter. Salts with 
organic basis such as amines can, however, also be used as starting substances. 

Methods for the activation of free or saiified carboxy groups are per se known, particularly in 
the field of peptide synthesis, and those skilled in the art can easily determine which method 

15 is the most suitable, especially whether or not to use the starting substances in their free or 
salified form. Activation methods per se known for peptide synthesis procedures and usefiil 
in the preparation procedures of the present invention are desoibed, for example, in 
Bodanszky, M., In search of new methods in peptide synthesis, Int. J. Peptide Protein Res. 25, 
1985, 449-474; and Gross, E. et al. The Peptides, Analysis Synthesis, Biology, Academic 

20 Press, Inc., 1979, Vol. 1, Chapter 2. According to such procedures, a carboxyl component is 
activated, that is, a carboxyl component is converted to a reactive form. Such aaivation 
typically involves a reaction between an acid and an activating agent according to the scheme: 

O 

II 

R-COOH ^ R-C-X 

wherein X is an electron Avithdrawing moiety. Most activated derivatives of carboxylic acids, 
25 therefore, are mbced anhydrides, including in the broad sense also acid azides and acid 
chlorides which can be considered mixed anhydrides of hydrazoic acid and HCl as the 



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activating agents. In addition, activation of a carboxyl component can be accomplished by 
the formation of intermediate "activated esters". These "activated esters" can be of various 
types, but particularly useful "activated esters" are those prepared by use of 
dicyclohexylcarbodiimide, p^nitrophenyl esters, trichlorophenyl esters, pentachlorophenyl 
5 esters, and 0-acyl derivatives of hydroxylamines, particularly esters of N- 
hydroxysuccinimide. 

All of these various types of activation procedures are useful in the preparation of the cross- 
linked HA of the invention, as all of these procedures can be characterized as importantly 
involving the reaction of a carboxyl group with an activating agent which essentially results in 
10 the formation of a substituent group that is easily reactive with a hydroxyl group so as to 

easily form the inner ester bonding characteristic of the products of the invention, the number 
of carboxy functions to be converted into inner esters in proportion to the number of activated 
carboxy functions and this number depends on the quality of the activating agent used. 

The preferred procedure for preparation of cross-linked HA is therefore charaaerized by 

1 5 treating HA, having free or salified carboxy groups, with an agent which activates the carboxy 
function, possibly in the presence of an auxiliary agent favoring the formation of intmnediate 
activated derivatives and/or a tertiary organic or inorganic base, exposing the mixture to 
heating or irradiation (particularly with UV light), and if desired, by salifying free carboxy 
groups or by freeing salified carboxy groups. Of the substances able to activate the carboxy 

20 group, the conventional ones described in literature can be used, for example, those usually 
used in the synthesis of peptides, except however those which would have the effect of 
ahering or destroying the molecular structure of the starting HA, such as those used for the 
formation of carboxyl halides. Preferred substances which lead to the formation of activated 
esters are those, aich as, carbodiimides, dicyclohexylcarbodiimide, benzyl- 

25 isopropylcarbodiimide, benzyl-ethyl-carbodiimide; ethoxyacetylene; Woodward's reagent 

ethyI-5-phenylisoxazolium-3-sulfonate) or halogen derivatives from aliphatic, cycloaliphatic 
or aromatic hydrocarbons, or from heterocyclic compound with halogen made mobile by the 
presence of one or more activating groups, such as chloroacetonitryl and especially the salts 
of 2-chloro-N-aIkylpyridine, such as chloride of 2-chloro-N-methyl-pyridine or other alkyl 

30 derivatives with inferior alkyl groups, such as those with up to 6 carbon atoms. In the place of 
chloride derivatives, other halogen derivatives can of course be used, such as bromide 
derivatives. 



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This activation reaction can be carried out in organic solvents, especially aprotic solvents such 
as dialkylsulfoxides, dialkylcarboxylamides, such as in particular lower alkyl 
dialkylsulfoxides, particularly dimethylsulfoxide, polymethylene sulfoxides, such as 
tetramethylene sulfoxide, dialkyls or polymethylene sulfones, such as tetramethylene sulfone, 
5 sulfolane and lower alkyl dialkyamides of lower aliphatic acids in which the alkyl groups 

have a maximum of six carbon atoms, such as dimethyl or diethyl formamide or dimethyl or 
diethyl acetamide. Other solvents may also be used, however, and these need not always be 
aprotic, such as alcohols, ethers, ketones, esters, such as lower aliphatic 
dialkyloxyhydrocarbides, such as dimethoxyethane and especially aliphatic or heterocyclic 

1 0 alcohols and ketones with a low boiling point, such as lower N-alkyl-pyrrolidones, such as N- 
methylpyrrolidone or N-ethyl-pyrrolidone, hexafluorisopropanol and trifluoroethanol. If 
halogen derivatives are used as carboxy I -activating substances, especially in the form of salts, 
such as the above-mentioned 2-chloro-N-methylpyridinium chloride, it is better to use a metal 
sah or a sak of the organic base of the starting polysaccharide, especially one of the 

1 5 quaternary ammonium saks described hereafter, such as tetrabutyl ammonium salt. These 
saks have the special advantage of being very soluble in the above said organic solvents in 
which the cross-linking reaction is best effected, thus guaranteeing an excellent yield. It is 
advisable to add to the mixture a substance capable of subtracting acid, such as organic bases, 
carbonates, bicarbonates or alkaline or alkaline earth acetates, or organic bases and especially 

20 tertiary bases such as pyridine and its homologues, such as coUidine, or aliphatic amine bas^ 
such as triethylamine or N-methyl-piperazine. 

The use of quaternary ammonium salts represents a particularly advantageous procedi^e- 
Such ammonium saks are well known and are prepared in the same way as other known saks. 
They derive from alkyls having preferably between 1 and 6 carbon atoms. It is preferable to 
25 use tetrabutyl ammonium saks. One variation in the procedure in which quaternary 
ammonium salts are used, consists in reacting an alkaline salt, for example, sodium or 
potassium salt, in the presence of catalyzing quantity of a quaternary ammonium salt, such as 
tetiabutylammonium iodide. 

The substances which catalyze artivation of the carboxy groups to be added to the activating 
30 agents are reported in lit^ature and these too are preferably bases such as those mentioned 

previously. Thus, for example, when the carboxy groups are activated wkh isothiazoline saks 
it is preferable to add some triethylamine to the reaction mixture. 



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The reaction of formation of activated intermediates, such as and especially esters, is carried 
out at the temperature recommended in literature and this temperature can, however, be varied 
should circumstances require as can be easily determined by one skilled in the art. The 
formation of inner ester bonds can come about within a fairly wide temperature range, for 
5 example between O"* and 1 50°, preferably room temperature or slightly above, for example 
between 20° and 75°. Raising the temperature favors the formation of inner ester bonds, as 
does exposure to radiations of suitable wavelength, such as ultraviolet rays. 

The substrate of hyaluronic acid can be of any origin, and can be of the various types 
discussed above. The preferred HA starting materials are those with an average molecular 
1 0 weight of 1 50,000 to 730,000, especially 1 50,000 to 450,000 daltons. 

In addition, the amount of internal cross-linking can vary, but preferred materials according to 
the invention utilize HA cross-linked to a degree of 3 to 15% of the carboxyl groups. 

When prepared in the form of gels, the cross-linked dervatives have greater viscosity than the 
unmodified hyaluronic acid. By controlling the viscosity, both the degradation time and 
1 5 effect on adhesion prevention can be varied. Preferred are those gels having a viscosity of at 
least 200 Pa'sec V More preferred are gels with a viscosity of at least 250 Pa'sec*' or even 
300 Pa'sec"' and most preferred are those gels having a viscosity of at least 350 Pa'sec** or 
400 Pa*sec*V 

The following Examples describe the preparation of useful cross-linked HA products for 
20 making the materials of the invention. 

Example 4 - Preparation of 3% Cross-Linked Hyaluronic Acid GJY) 
Product description: 

3% of carboxy groups used in internal esterification. 

97% of carboxy groups salified with sodium. 

25 6.21 g of HA tetrabutylammonium salt with a molecular weight of 170,000 corresponding to 
10 mEq of a monomeric unit are solubilized in 248 ml of DMSO at 25°C, 0.03 g (0.3 rrEq) of 
triethylamine are added. 



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A solution of 0.076 g (0.3 mEq) of 2-chloro-l -methyl pyridinium chloride in 60 ml of DMSO 
is slowly added drop by drop over a time interval of 1 hour and the mixture is kept for 15 
hours at 30°C- 

A solution formed by 100 ml of water and 2.5 g of sodium chloride is then added and the 
5 resulting mixture is then poured slowly into 750 ml of acetone, maintaining continual 

agitation. A precipitate is formed which is then filtered and washed three times in 100 ml of 
acetone water 5:1 and three times with 100 ml of acetone and lastly vacuum-dried for 24 
hours at 30°. 

4 g of the title compound are obtained. Quantitative determination of the ester groups is 
1 0 carried out according to the saponification method described on pp. 169-172 of "Quantitative 
Organic Analysis Via Functional Groups", 4th Edition, John Wiley and Sons Publication. 

Example 5 - Preparation of 5% Cross-Linked Hyaluronic Acid f ACP 5%) 
Product description: 

5% of carboxy groups used in internal esterification. 

1 5 95% of carboxy groups salified with sodium. 

6.21 g of HA tetrabutylammonium salt with a molecular weight of 95,000 corresponding to 
10 mEq of a monomeric unit are solubilized in 248 ml of DMSO at 25X, 0.05 1 gr (0.5 mEq) 
of triethylamine are added and the resulting solution is ^itated for 30 minutes. 

A solution of 0,128 gr (0.5 mEq) of 2-chloro-l -methyl pyridinium iodide in 60 ml of DMSO 
20 is slowly added drop by drop over a time interval of 1 hour and the mixture is kept for 15 
hours at 30°C 

A solution formed by 100 ml of water and 2.5 gr of sodium chloride is then added and the 
resulting mixture is then poured slowly into 750 ml of acetone, maintaining continual 
agitatioa A precipitate is formed which is then filto-ed and washed three times in 100 ml of 
25 acetone water 5:1 and three times vnth 100 ml of acetone and lastly vacuum-dried for 24 
hours at 30^ 



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3.95 grs of the title compound are obtained. Quantitative determination of the ester groups is 
carried out according to the saponification method described on pp. 169-172 of "Quantitative 
Organic Analysis Via Functional Groups", 4th Edition, John Wiley and Sons Publication. 

5 Example 6 - Preparation of 10% Cross-linked Hyaluronic Acid (HY) 
Product description: 

10% of carboxy groups used in internal esterifi cation. 
90% of carboxy groups salified with sodium. 

6.21 g of HA tetrabutylammonium salt with a molecular weight of 620,000 corresponding to 
10 10 mEq of a monomeric unit are solubilized in 248 ml of DMSO at 25°C. 0.101 gr (1.0 mEq) 
of triethylamine is added and the resulting solution is agitated for 30 minutes. 

A solution of 0.255 gr ( 1 .0 mEq) of 2-chloro-l-methyl-pyridinium chloride in 60 ml of 
DMSO is slowly added drop by drop over a time interval of 1 hour and the mixture is kept for 
15 hours at 30X. 

15 A solution formed by 1 00 ml of water and 2.5 gr of sodium chloride is then added and the 
resulting mixture is then poured slowly into 750 ml of acetone, maintaining continual 
agitation. A precipitate is formed when is then filtered and washed three times in 100 ml of 
acetone water 5 : 1 and three times with 1 00 ml of acetone and lastly vacuum-dried for 24 
hours at 30^ 

20 3.93 grs of the title compound are obtained. Quantitative determine of the ester groups is 

carried out according to the saponification method described on pp. Ii69-172 of "Quantitative 
Organic Analysis Via Functional Groups", 4th Edition, John Wiley and Sons Publication. 

Example 7 - Preparation of 1 5% Cross-liriced Hvaluronic Acid (HY) 
25 Product Description: 

15% of carboxy groups used in internal esterification. 



85% of carboxy groups salified with sodium. 



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-17. 

6.2rgr of HA tetrabutylammonium salt with a molecular weight of 170,000 corresponding to 
10 mEq of a monomeric unit are solubilized in 248 ml of DMSO at 25°C, 0. 152 gr (L5 mEq) 
of triethylamine chloride are added and the resulting solution is agitated for 30 minutes. 

A solution of 0.382 g (1.5 mEq) of 2-chloro-l-methyl-pyridinium-chloride in 20 ml of DMSO 
5 is slowly added drop by drop over a time interval of 1 hour and the mixture is kept at a 
temperature of 30° for 45 hours. 

A solution made up of 100 ml of water and 2.5 of sodium chloride is added and the resulting 
mixture is slowly poured into 750 mi of acetone, maintaining continual agitation, A 
precipitate is formed which is then filtered and washed three times with 100 ml of 
1 0 acetone/H20 5: 1 and three times with 100 ml of acetone finally vacuum^iried for 24 hours at 
a temperature of 30°. 

3.9 g of the title compound are obtained Quantitative determination of the total ester groups 
is carried out according to the saponification method described on pp, 169-172 of 
"Quantitative Organic Analysis Via Functional Groups", 4th Edition, John Wiley and Sons 
1 5 Publication. 

4. The Mammalian Cell and/or Molecular Component 

The compositions of the invention are particularly usefiil in providing an optimal delivery 
system for the local application of cells and/or biological and/or pharmacological 
molecules. Many pathologies are due to the significant loss of substance which is barely 

20 self-repaired by natural host-driven mechanisms or even not repaired at all. In almost all 

cases, the repair yields a tissue not equal, in terms of biological, histological and functional 
characteristics, of the original undamaged tissue. In this regard. Tissue Engineering, that is 
the combination of cells embedded or layered onto a biocompatible scaffold, offers now 
the possibility to build in vitro a tissue-like structure which can undergo further 

25 maturation/differentiation once grafted into the patient with the potential to completely 
regenerate the original lost tissue (ref : Langer and Vacanti, Science, 1993). 

In other pathologies, the ability x)f tissue/organ to function property or to recover fi-om a 
specific disease relies on the application of certain biologically active molecules, such as, 
for instance, growth factors (such as those per se known in the art), or pharmacological 
30 substances, such as, for instance, antibiotics which are known in the art. The major 



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difficulty in applying such a drug-based therapy is the correct delivery of said molecules 
which must reach the specific target and act within a specific window in order to maximize 
the expected curative effect and, in same cases, decrease the toxic potential toward other 
tissues/organs. 

5 Yet other pathologies may require a much more complex treatment approach. In particular, 
specific diseases, such as, for instance, dysmetabolic conditions, need not only a local 
delivery of curative drug, but also a bio-interactive control in the administration of the 
substance. One paradigmatic example is constituted by the treatment of insulin-dependent 
diabetes (type I). The success of any therapeutic protocol is based on the administration of 

1 0 insulin only when haematic glucose levels reach specific values. In this case, only a 

biological sensible system making insulin may appropriately respond to the body needs. In 
the particular case, since pancreatic islet cells are actually not easily manipulable with cell 
culture technology, other cell types, for instance fibroblasts, can be genetically modified in 
order to express insulin in a regulated fashion. For these specific diseases, the ideal clinical 

1 5 protocol should be constituted by the local delivery of cells, previously committed to 

produce the specific biological/pharmacological molecule, in a suitable carrier. The cells 
deliverable by the present invention are mammalian cells, especially those selected firom 
the group consisting of chondrocytes, osteocytes, fibroblasts, keratinocytes, adipocytes, 
muscle cells, nerve cells, cells fi-om the peripheral nervous system, endothelial cells, 

20 hematopoietic cells, glandular cells, cells of the urethra and stem cells, both fi-om adult and 
embryonic tissue. 

For example, the chondrogenic cells may be isolated directly from pre-existing cartilage 

tissue, for example, hyaline cartilage, elastic cartilage, or fibrocartilage. Specifically. 

chondrogenic cells may be isolated from articular cartilage (fi-om either weight bearing or 
25 non-weight bearing joints), costal cartilage, nasal cartilage, auricular t:artilage, tracheal 

cartilage, epiglottic cartilage, thyroid cartilage, arytenoid cartilage and cricoid cartilage. 

Methods for isolating chondrogenic cells fi-om such tissues are set forth hereinbelow. 

Alternatively, chondrogenic cells may be isolated fi-om bone marrow. See for example, 

U.S. Pat. Nos. 5,197,985 and 4,642,120, and Wakitani et al. (1994) J. Bone Joint Surg. 
30 76:579-591, the disclosures of which are incorporated by reference herein. 



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Once chondrogenic cells have been isolated from the pre-existing tissue they are 
proliferated ex vivo in monolayer culture using conventional techniques well known in the 
art. See for example, Pollack (1975) in "Readings in Mammalian Cell Culture", Cold 
Spring Harbor Laboratory Press, Cold Spring Harbor, the disclosure of which is 
5 incorporated by reference herein. Briefly, the population of chondrogenic cells is expanded 
by culturing the cells as monolayers and by serially passaging the cells. The chondrogenic 
cells are passaged after the cells have proliferated to such a density that they contact one 
another on the surface of the cell culture plate. During the passaging step, the cells are 
released from the substratum. This is performed routinely by pouring a solution containing 

10 a proteolytic enzyme, i e, trypsin, onto the monolayer. The proteolytic enzyme hydrolyzes 
proteins which anchor the cells on the substratum. As a result, the cells are released from 
the surface of the substratum. The resulting cells, now in suspension, are diluted with 
culture medium and replated into a new tissue culture dish at a cell density such that the 
cells do not contact one another. The cells subsequently reattach onto the surface of the 

1 5 tissue culture and start to proliferate once again. Alternatively, the cells in suspension may 
be cryopreserved for subsequent use using techniques well known in the art. See for 
example. Pollack (supra). 

The cells are repeatedly passaged until enough cells have been propagated to prepare a 
piece of synthetic cartilage of pre-determined size. As a result, a population containing a 
20 small number of chondrogenic cells originally isolated from a biopsy sample may be 

expanded in vitro thereby to generate a large number of chondrogenic cells for subsequent 
use in the practice of the invention. 

In another preferred embodiment, polypeptide growth factors may be added to the 
chondrogenic cells in the pre-shaped well to enhance or stimulate the production of 

25 cartilage specific proteoglycans and/or collagen. Preferred growth factors include, but are 
not limited to, transforming growth factor-beta (TGF-.beta.), insulin-iike growth factor 
(IGF), platelet derived growth factor (PDGF), epidermal growth factor ^GF), acidic or 
basic fibroblast growth factor (aFBF or bFBF), hepatocytic growth factor (HGF), 
keraiinocyte growth factor (KGF) the bone morphogenic factors (BMPs) including; BMP- 

30 1; BMP-2; BMP-3; BMP-4; BMP-5; and BMP-6 and the osteogenic proteins (OPs) 

including: OP-1; OP-2; and OP-3. In addition, it is contemplated that ascorbate may be 
added to the chondrogenic cells in the pre-shaped well to enhance or stimulate the 



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production of cartilage specific proteoglycans and collagen. However, these particular 
compounds are not limiting. Any compound or composition capable of stimulating or 
inducing the production of cartilage specific proteoglycans and collagen may be useful in 
the practice of the instant invention. 

5 4.1 Procedures for Isolation of Chondrocytes 

Briefly, tissue containing chondrogenic cells is disaggregated to release denuded 
chondrogenic cells from their extracellular matrix. The denuded cells then are isolated and 
proliferated as monolayers in an undifferentiated state ex vivo. The passaging procedure 
may be repeated multiple times (n), for example up to about 7 to 10 passages until enough 
1 0 cells have been propagated to prepare a piece of cartilage of pre-determined size. These 
steps expand the number of chondrogenic cells in a population that can be used 
subsequently to form the three-dimensional, multi cell-layered patch of synthetic cartilage. 

The proliferated but undifferentiated chondrogenic cells then are seeded into a pre-shaped 
well having a cell contacting, cell adhesive surface. The cell abhesive surface prevents 

1 5 chondrogenic cells cukured in the well from attaching to the surface of the well. The cells, 
deprived of anchorage, interact with one another and coalesce within hours to generate a 
cohesive plug of cells. The chondrogenic cells then begin to differentiate, as characterized 
by the production and secretion of cartilage-specific markers, i.e., type II collagen and 
sulfated proteoglycans. Type II collagen is found specifically in cartilage. The 

20 chondrogenic cells then are cultured in the well for time sufficient to permit the formation 
of a three-dimensional, multi cell-layered patch of synthetic cartilage. The resulting 
synthetic cartilage patch comprises chondrogenic cells dispersed with a new, endogenously 
produced and secreted extracellular matrix. The extracellular matrix deposited during this 
procedure is biochemically and morphologically similar to the extracellular matrix found 

25 in natural cartilage. Specifically, the synthetic matrix comprises fibers of type II collagen, 
and sulfated proteoglycans such as chondroitin sulfate and keratan sulfate. 

4.2 Isolation of Tissue Containing Chondrogenic Cells 

Chondrogenic cells useful in the practice of the instant invention may be sampled from a 
variety of sources in a mammal that contain such cells, for example, pre-existing cartilage 
30 tissue, perichondrial tissue or bone marrow. 



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Although costal cartilage, nasal cartilage, auricular cartilage, tracheal cartilage, epiglottic 
cartilage, thyroid cartilage, arytenoid cartilage and cricoid cartilage are useful sources of 
chondrogenic cells, articular cartilage (from either weight bearing or non-weight bearing 
joints) is the preferred source. Biopsy samples of articular cartilage may be readily isolated 
by a surgeon performing arthroscopic or open joint surgery. Procedures for isolating 
biopsy tissues are well known in the art and so are not described in detailed herein. See for 
example, "Operative Arthroscopy" (1991) by McGinty et al.,; Raven Press, New York, the 
disclosure of which is incorporated by reference herein. 

Perichondrial tissue is the membranous tissue that coats the surface of all types of 
cartilage, except for articular canilage. Perichondrial tissue provides nutrients to the 
chondrocytes located in the underlying unvascularized cartilage tissue. Perichondrial tissue 
sampled from costal (rib) cartilage of patients suffering from osteoporosis provides a 
source of chondrogenic cells when the normal articular cartilage is diseased or unavailable. 
Biopsy samples of perichondrial tissue may be isolated from the surface of costal cartilage 
or alternatively from the surface of auricular cartilage, nasal cartilage and cricoid cartilage 
using simple surgical procedures well known in the art. See for example: Skoog et al. 
(1990) Scan. J. Plast. Reconstr. Hand Surg. 24:89-93; Bulstra et al. (1990) J. Orthro. Res. 
8:328-335; and Homminga et al. (1990) J. Bone Constr. Surg. 72:1003-1007, the 
disclosures of which are incorporated by reference herein. 

It is contemplated also that chondrogenic cells, specifically mesenchymal cells, useful in 
the practice of the invention may be isolated from bone marrow. Surgical procedures 
useful in the isolation of bone marrow are well known in the art and so are not described in 
detailed herein. See for example, Wakitani et al. (1994) J. Bone Joint Surg. 76: 579-591, 
the disclosure of which is incorporated by reference herein. 

4.3. Preparation of Denuded Chondrogenic Cells 

Protocols for preparing denuded chondrogenic cells from cartilage tissue, perichondrial 
tissue, and bone marrow are set forth below. 

A. From Articular Cartilage 
Articular cartilage, both loaded (weight bearing) and unloaded (non-weight bearing), may 
be subjected to enzymatic treatment in order to disaggregate the tissue and release denuded 
chondrogenic cells from the extracellular matrix. Solutions containing proteolytic 



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enzymes, for example, chondroitinase ABC, hyaluronidase, pronase, collagense, or trypsin 
may be added to articular cartilage tissue in order to digest the extracellular matrix. See for 
example. Watt & Dudhia (1988) Differentiation 38: 140-147, the disclosure of which is 
incorporated herein by reference. 

5 In a preferred procedure, articular cartilage is initially cut into pieces of about 1 mm in 
diameter, or less. This is routinely performed using a sterile scalpel. The minced tissue 
then is disaggregated enzymatically, for example, by the addition of a solution containing 
0.1% collagenase (Boehringer Mannheim GmbH, Germany). Approximately 1 ml of 
collagenase is added per 0.25 ml equivalents of minced tissue. The sample is then mixed 

10 and incubated overnight (up to 16 hours) at 37.degree. C, v^^ith agitation. Following the 
overnight digestion, the residual pieces of tissue are harvested by centrifugation, the 
supernatant removed, and the remaining cartilage pieces redigested by the addition of a 
solution containing, for example, 0.25% collagenase and 0.05% trypsin (Sigma Chemical 
Co., St. Louis). Approximately 1 mi of 0.25% collagenase, 0.05% trypsin is added per 0.25 

15 ml equivalents of residual tissue. The sample then is mixed and incubated for 2-4 hours at 
37.degree. C, with agitation. Any remaining tissue is pelleted by centrifugation and the 
cell suspension harvested. The collagenase, trypsin step is repeated 2-4 times or until the 
tissue is completely disaggregated 

The enzymatic reaction is terminated by the addition of tissue culture medium 
20 supplemented with approximately 10% fetal bovine serum (FBS) (Hyclone, Logan, Utah). 
A preferred cell culture medium includes, for example, Dulbecco's minimal essential 
medium (DMEM) (Sigma Chemical Co., St. Louis) supplemented with 10% FBS. An 
alternative cell culture medium includes a 1:1 (vol/vol) mixture of Medium 199 (Sigma 
Chemical Co., St. Louis) and Molecular Cell Developmental Biology Medium 202 
25 (MCDB 202) (Sigma Chemical Co., St. Louis), respectively, supplemented with 10% FBS. 
Alternatively, another cell culture medium useful in the practice of the invention includes a 
3:1 (vol/vol) mixture of DMEM and Ham's F-12 (F12) (Sigma Chemical Co., St. Louis), 
respectively, supplemented with 10% FBS. Fractions containing denuded chondrogenic 
cells are combined, and the cells inoculated into a cell culture dish at a plating density of 
30 about Ix 10^ - 5 X lO' cells/cm^ preferably about 5 x 10^ - 1 x lO' cells/cm^, and most 
preferably about 1 x 10^ - 1 x lO"* cells/cm^, for cell expansion and testing. 



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Chondrocytes may be isolated from costal cartilage, nasal cartilage, auricular cartilage, 
tracheal cartilage, epiglottic cartilage, thyroid cartilage, arytenoid cartilage and cricoid 
cartilage using the aforementioned procedure. 

B. From Perichondrial Tissue 

Denuded chondrogenic cells preferably are isolated from perichondrial tissue using the 
same procedure as described in section II A, hereinabove. 

Briefly, the tissue is minced into pieces of about 1 mm in diameter, or less. The minced 
tissue is repeatedly digested with proteolytic enzymes, for example, trypsin and 
collagenase. The resulting denuded cells are inoculated into a cell culture dish at a plating 
density of about 1 x 10^ - 5 x 10^ cells/cm^, preferably about 5 x 10^ to 1 x 10^ cells/cm^, 
and most preferably about 1 x 10^ - 1 x 10^ cells/cm^ for cell expansion and testing. 

Alternatively, a non-destructive procedure may be used to isolate chondrogenic cells from 
perichondrial tissue. In this procedure, intact explant tissue is placed in a cell culture dish 
and incubated in growth medium. The chondrogenic cells located within the tissue migrate 
out of the tissue and onto the surface of the tissue plate where they begin to proliferate. See 
for example, Bulstra et al. (1990) J. Orthop. Res. 8:328-335, the disclosure of which is 
incorporated by reference herein. Briefly, pieces of the minced explant tissue are placed 
into a tissue culture plate containing tissue culture medium, A preferred cell culture 
medium comprises DMEM supplemented with 10% FBS. The explant tissues are 
incubated at 37X., 5% CO2 for 3-7 days. During this time the chondrogenic cells migrate 
out of the explant tissue and onto the surface of the tissue culture dish. After reattaching to 
the surface of the plate, the cells start to proliferate again. 

C. From Borie Marrow 

Chondrogenic cells, specifically mesenchymal cells, may be isolated from samples of bone 
marrow. Procedures usefijl for the isolation of mesenchymal cells from bone marrow are 
well known in the art, see for example: U.S. Pat. Nos. 5,197,985; 4,642,120; and Wakitani 
et al. (1994, supra). 

For example, in a preferred method, a plug of bone marrow may be removed surgically 
from the mammal of interest and added to cell culture medium. Preferred comi^ete growth 
media are disclosed in U.S. Pat. No. 5,197,985. The mixture then is vortexed to break up 



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the plug of tissue. The resulting suspension is centrifuged to separate bone marrow cells 
from large pieces of particulate matter i.e., bone fragments. The ceils then are dissociated 
to give a single cell suspension by forcing the cells through a syringe fitted with a series of 
16, 18, and 20 gauge needles. The cells then are plated out into a tissue culture plate at a 
5 cell density of about 1 x 10^ - 1 x 10^ cells/cm^ for selectively separating and/or isolating 
bone marrow derived mesenchymal cells from the remaining cells present in the 
suspension. 

III. Expansion of Denuded Chondrogenic Cells In Vitro Chondrogenic cells isolated from 
cartilage tissue, perichondrial tissue, or bone marrow using the methods described in 

10 seaion II hereinabove may be placed in monolayer culture for proliferative expansion. The 
process enables one to amplify the number of isolated chondrogenic cells. In principal, the 
artisan may produce essentially an unlimited number of chondrogenic cells and therefore 
essentially an unlimited amount of synthetic cartilage. It is appreciated, however, that 
during proliferative expansion the chondrogenic cells dedifferentiate and lose their ability 

15 to secrete cartilage specific extracellular matrix. A procedure to assay whether the 

undifferentiated cells still retain their chondrogenic potential is described ho-einbelow. 

4A Cell Proliferation 

Protocols for proliferating cells by monolayer culture are well known in the art, see for 
example, Pollack (supra), and so are not described in detail herein. 

20 Briefly, monolayer cultures are initiated by inoculating primary chondrogenic cells, 
isolated from either cartilage tissue or perichondrial tissue, into a cell culture dish at a 
plating density density of about 1 x 10^ - 5 x 10^ cells/cm^, more preferably about 5x10^ 
- 1 x lO' cells/cm^ and most preferably about 1 x 10^ - 1 x 10** cells/cm^. Chondrogenic 
cells that have undergone one or more cycles of passaging are also plated out at the same 

25 plating densities. Primary chondrogenic cells isolated from bone marrow are plated out 
into a tissue culture plate at a cell density of about 1 x 10^ - 1 x 10^ cells/cm^. 
Chondrogenic cells from bone marrow that have undergone one or more cycles of 
passaging are plated out at plating densities of about 1 x 10^ - 5 x 10^ cells/cm^, more 
preferably about 5 x 10^ - 1 x 10^ cells/cm^ and most preferably about 1 x 10^ - 1 x 10^ 

30 cells/cm^. The chondrogenic cells subsequently are cultured at 37°C., 5% CO2 ini:ell 
culture medium. 



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A preferred cell culture medium comprises DMEM supplemented with 10% FBS. 
Alternatively, a cell culture medium comprising a 1:1 (vol/vol) mixture of Medium 199 
and MCDB 202, respectively, supplemented with 10% FBS may be used. Still another cell 
culture medium useful in the practice of the invention comprises a 3: 1 (vol/vol) mixture of 
5 DMEM and Fl 2, respectively, supplemented with 1 0% FBS. 

Once the cell cultures become confluent, i.e., the cells grow to such a density on the 
surface of the plate that they contact one another, the cells are passaged and inoculated into 
a new plate. This may be accomplished by initially removing the cell culture medium 
overlaying the cells monolayer by aspiration, and washing the cell monolayer with 

10 phosphate buffered saline (PBS). The PBS is removed, by aspiration, and a solution 

containing a proteolytic enzyme, i.e., 0.1% trypsin, then is poured onto the monolayer. The 
proteolytic enzyme hydrolyzes proteins that anchor the cells onto the surface of the plate 
thereby releasing the cells from the surface of the plate. The proteolytic enzyme in the cell 
suspension then is inactivated by adding FBS to the suspension to give a final 

1 5 concentration of 10% (vol/vol). The density of cells in the suspension then is estimated and 
the cells re-plated into a new cell culture plate at a density of about 1 x 10^ - 5 10^ cells, 
more preferably about 5 x 10^ -1 x 10^ cells, and most preferably about 1 x 10^ - 10^ cells 
per cm^. The passaging procedure may be repeated muhiple times, for example up to about 
7 to 10 times, until enough cells have been propagated to prepare a piece of cartilage of 

20 pre-determined size. 

It is appreciated that suspensions of proliferated cells may be cryopreserved indefinitely 
using techniques well known in the art. See for example. Pollack (supra). Accordingly, 
populations of chondrogenic cells may be stored for subsequent use whenever a necessity 
arises. 

25 4.5 Assay To Measure Chondrogenic Potential of Proliferated Cells 

Undifferentiated chondrogenic cells, expanded in monolayer culture, may be assayed to 
detmnine whether they still retain their chondrogenic potential. This may be performed by 
culturing the cells in a semi-solid medium in a process called agarose culture. This 
procedure is described in Benya et al. ( 1 982) Cell 30:21 5-224, the disclosure of which is 
30 mcorporated by reference herein. 



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Briefly, proliferated chondrogenic cells are seeded into a solution of warm 2% low melting 
temperature agarose (LT agarose) (BioRad, Richmond, Calif). The use of LT agarose 
permits cells to be seeded into the agarose without thermal damage to the cells. The 
agarose is cooled to about 39-4 1°C. prior to the addition of cells. Approximately 1x10^- 
5 1x10^ cells are seeded into 1 ml of the liquid agarose. 

The cells are cultured subsequently at 37°C., 5% CO2 for 3-4 weeks in a cell culture 
medium preferably containing DMEM supplemented with 10% FBS. During this time, the 
chondrogenic cells replicate to from colonies which start to secrete an extracellular matrix. 
The resulting colonies have the appearance of small "nodules" embedded within the 
1 0 agarose. The colonies may be counted and the chondrogenic proportion of cells determined 
histochemically and immunohistochemically using procedures well known in the art. 

4.6 Preparation Of Cell Cultures From Bone Marrow Stroma 

Bone marrow stroma can be isolated by aspiration from the iliac crest in sterile conditions 
and according to standard procedures, by means of a heparin-treated plastic tube connected 

15 to a 10-ml syringe containing 1 ml of heparin solution (3,000 units/ml). Besides bone 

marrow itself, it is possible to use stem cells isolated from bone marrow. In this case the 
medial proximal surface of the tibia (or any other bone) is exposed under anaesthetic 
through a small incision. The subcutaneous tissue and the periosteum are incised and 
folded back to expose the bone surface. The tibia is perforated with a 16- or 18-gauge 

20 needle and the bone marrow is aspirated through a heparin-treated plastic tube attached to a 
syringe containing I ml of a heparin solution (3,000 units/ml). The aspirated matto" is 
transferred, under sterile conditions, into a 50-ml plastic tube and centrifuged for 10 
minutes at 1,300 rpm. The centrifuged cells are washed thr^e times with warm Hank*s 
basic saline solution (HBSS), centrifuged again and suspended in a complete culture 

25 medium containing alpha minimum essential medium (a-MEM) enriched with a 1% 
antibiotic solution (10,000 units of penicillin, 10 mg/ml of streptomycin), 10% foetal 
bovine serum (FBS), basic fibroblast growth factor (bFGF) (10 ng/ml) and ± 
dexamethasone (0.4 jig/ml). The cell suspension is poured into a 35-mm Petri capsule at a 
density of 3-5 x 10* nucleate cells per cm^. The mesenchymal stem cells are incubated in a 

30 complete culture medium at 37*C in a humidified atmosphere containing 5% CO2 and 95% 
air. 



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After four days of primary culture the undifferentiated cells are removed by washing with a 
phosphate buffer solution. The culture medium is changed every three days. 

When the cells reach confluence after about 2-3 weeks, they are removed from the culture 
container by enzymatic digestion for 7 minutes at 37°C with trypsin 0.05%, EDTA 0.02%. 
5 The reaction is interrupted by the addition of complete culture medium, the cell suspension 
is transferred to a 50-ml plastic tube and centrifuged for 10 minutes at 1,400 rpm. The cells 
are resuspended in a culture medium and counted with a haemocytometer. 

In order to induce chondrogenesis in the mesenchymal stem cells and bone marrow cells, 
the mass culture technique is used (initial cell density > 1 x 10^ cells/cm^). 

1 0 4.7 Preparation of cell cultures from cartilage tissue 

A biopsy of joint cartilage is taken by standard surgical procedures. 

The specimen of cartilage is disintegrated by enzymatic digestion using a solution of 0.1% 
coUagenase. Approximately 1 ml of collagenase per 0.25 ml of minced tissue is added. The 
specimen is mixed and incubated for about 16 hours at 37*'C under agitation. Subsequently 
1 5 the fragments of residue tissue are separated by centrifiigation and the supernatant is 

removed. The fragments of remaining cartilage are exposed to enzymatic digestion again in 
a solution containing 0.25% collagenase and 0.05% trypsin. The specimen is mixed and 
incubated for 2-4 hours at 37'*C under agitation. The remaining tissue is separated by 
centrifiigation and the treatment is repeated until digestion is complete. 

20 The enzymatic reaction is intermpted by the addition of a culture medium eiiriched with 
10% foetal bovine serum (FBS) or with Dulbecco's minimal essential culture medium 
enriched with 1 0% FBS. 

The cell suspension is poured into a 35-mm Petri dish at a density of 3-5 x 10* cells per 
cm^. 

25 5. The Pharmaceuticallv or Biologically Active Component 

Since it has been found that the 50-70% benzyl est^r of HA and the 3-15% ACP HA 
derivatives are excellent carries for a delivery system of injectable administration, the 
biologically or pharmacologically active componwit can be of any type tlesir-ed to be 
adnunistered to a mammal, such as a human patient. Of particular importance as 



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pharmacologically active substances are antibiotics, anti-inflammatory agents, antiseptics, 
active hormones, anti-tumoral agents, and anti-viral agents which are per se known to 
those in the art. 

The biologically active substances are preferably those which have an effect on the 
5 biological process of the mammal or patient. Of particular importance are substances 

which favor the adhesion of cells to the biomaterial, such as fibronectin, RGD or integrin 
sequences, the growth factors such as transforming growth factor P (TGPP), insulin-like 
growth factor (IGF), platelet-derived growth factors (PDGF), epidermal grov^h factors 
(EGF), acid or basic fibroblast growlh factors (aFBF or bFBF), hepatocyte growth factor 
1 0 (HGF), keratinocyte growth factor (KGF), bone morphogenic proteins (BMPS) such as 

BMP- 1, BMP-2, BMP-3, BMP-4, BMP-5 and BMP-6 and osteogenic proteins (OPs) such 
as OP-1, OP-2 and OP-3, the nucleic acids encoding specific genes or gene sequences or 
gene transcripts such as DNA and RNA, and differentiation/modulation factors. 

6. Additional Components 

1 5 The compositions of the invention are prepared in the form of a gel containing at least one 
of the benzyl ester or ACP derivatives and at least one biologically or pharmacologically 
active component and/or mammalian cell. The gel can also contain one or more derivatives 
of hyaluronic acid in one or more of various forms such as fibers, granules, microspheres, 
nanospheres, fragments of sponge. These forms can provide anchorage for the mammalian 

20 cells of the composition and are preferably comprised of the total benzyl ester hyaluronic 
acid derivative (HYAFF-1 1). The forms can preferably l)e made by the following 
procedures. 

The microspheres are preferentially prepared by the process described in EPOS 17565. The 
nanospheres are preferentially prepared by the process described in WO 96/29998. The 
25 sponges are preferentially prepared by the process described in U.S. Patent No. 4,851,521. 
The fibres can be prepared according to the procedures described in U.S. patents 5,520,916 
and 5,824,335. 

Example 8 - Microspheres 

A total benzyl ester hyaluronic acid derivative, where all the carboxy groups of HYAFF- 
30 1 1, as described in U.S. P^ent No. 4,85 1,521 is dissolved in a an aprotic solvent such as 
dimethylsulfoxide, at a concentration varying between 5 and 10% weight/volume. 



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generally 7% w/v. Once the polymer has solubilized, the mixture obtained will be referred 
to hereinafter as the disontinuous phase. At the same time, a mixture is prepared in a 
suitable reactor of high- viscosity mineral oil containing Arlacel^, a non-ionic surface- 
active agent, at a concentration of 1% w/v. 

5 This mixture will be referred to hereinafter as the continuous phase. 

The continuous phase is kept at IS'^C while being stirred at a fixed speed of 1000 RPM, 
then the discontinuous phase, prepared as previously described, is added to it. In these 
conditions, emulsification of he two phases is instantaneous. The ratio between the two 
phases (discontinuous and continuous) is about 1 to 16. 

1 0 After 1 5 minutes of stirring, acetylacetate is added. This solvent mixes perfectly with the 
two phases of the emulsion but it is a nonsolvent for the polymer and the human insulin 
polypeptide. It has been proven that the volume of extracting solvent needed to obtain 
complete extraction is two and a half times the total volume of emulsion. To facilitate 
extraction the stirring speed is set at 1400-1 500 RPM for 10 minutes and then lowered to 

1 5 500 RPM. The suspension thus obtained continues to be stirred while being pumped with a 
screw pump through a filter press set at 1 atmosphere. Once this filtration is complete, it is 
pumped through a filter of normal-hexane, this being a solvent with the double action of 
"drying'' the preparation and solubilizing any residue surfactant which may be present on 
the surface of the microspheres. The product is then put in suitable containers and stored at 

20 

In these working conditions the resulting mean particle size is 10 ^m. 

,7. Exemplary Compositions According to the Invention 

The following represent examples of the composition according to the invention. 



25 



Example 9 - Composition of gel of autocross-linked hvaluronic acid f ACP) containing 
fi-agments of autocross-linked hyaluronic acid (AC?) or total benzvl ester fHYAFF-1 1) 
sponge and cells 



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A sponge of ACP (or HYAFF- 1 1 ) is brought to a temperature of less than -1 50'C in liquid 
nitrogen, pressed and sieved to obtain a granulometry of less than 100 micron. One 
hundred mg of granules of ACP is mixed with 0,5 ml of ACP. 

Five to ten ml of heparin-treated bone marrow is transferred into a sterile syringe (22 
5 gauge) from 10-20 ml containing ACP granules and gel. The mixture is extruded slowly 
into a second syringe so as to obtain a homogeneous mixture. 

The cells can be injected w vivo into the osteochondral defect immediately afterwards or 
left to adhere to the microparticles for 3-4 hours at 37°C before implant. 

When the cells are previously expanded in vitro for a certain length of time (2-3 weeks), a 
1 0 knowm number of cells are suspended in a certain volume before mixing the culture with 
the gel. The volume of the suspension is calculated so as to avoid excessive dilution of the 
gel. 

An alternative method consists in mixing about 1-2 ml of bone marrow and mesenchymal 
stem cells with a mixture constituted by 100 mg of fragments of sponge and 35 mg of ACP 
1 5 powder inside a sterile syringe (22 gauge). The mixture is kept at 37°C for 3-4 hours so as 
to allow the powder to become hydrated and the cells to adhere to the fragments of sponge. 

The following Examples describe the preparation and administration of various 
combinations of the Hyaluronic Acid component previously described and the mammalian 
cell and/or molecular component. These examples will be applicable to both soft, such as 
20 skin, liver, intestine, and hard, bone and cartilage, tissues. 

Example 10 - Treatment of chondral and osteochondral defects with chondrogenic cells 
embedded in an injectable hvaluronate acid derivative-based gel 

The intended composition can be made in the form of an injectable gel containing at least 
one hyaluronic acid derivative wherein the chondrogenic cells are evenly dispersed. The 
25 gel can also contain one or more derivatives of hyaluronic acid in various forms such as 
fibers, granules, miaospheres, nanospheres, fragments of sponge, etc. Firstly, the 
chondrogenic cells, such as, for instance, adult differentiated chondrocytes or 
undifferentiated mesenchymal stem cells, are harvested from original tissues, that are, for 
instance, non-weight-bearing articular cartilage and bone marrow stroma. Cells are isolated 



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and expanded with standard cell culture techniques routinely used by whom skilled in the 
art. When a suitable cell number, based on the defect size and depth, is achieved, cells are 
detached from cuhure bidimensional surfaces and embedded in a gel constituted by ACP 
or HYAFF partial esterified. The relative cross-linking or esterification rate of the HA- 
5 based delivery vehicle may vary according to the desired degradation time to be achieved 
in the patient. Examples of the preparation of the benzyl esters of HA and autocross-linked 
HA derivatives have been reported previously. 

The gel can be manipulated mechanically in such a way that cells result evenly dispersed 
in the carrier. Then, the combination is injected by the use of a sterile syringe and/or 

10 arthroscopic device, routinely used by surgeons skilled in the art, to fill the defect size. Due 
to the property of HA-carrier, cells stay in the defect and start to make an extracellular 
matrbc that will substitute the carrier in the repair/regeneration period. Moreover, 
differentiation and/or growth factors may be added to the delivered combination of cells 
and gel in order to committ undifferentiated chondrogenic cells, for instance when using 

15 mesenchymal stem cells, or to stimulate the growth of administered cells and/or host cells. 

Example 1 1 - Treatment of chondral and osteochondral defects with chondrogenic cells 
embedded in injectable hvaluronic acid derivatives solid/gel formulations 
In order to inject a composition of cells which may have made some extracellular matrix 
molecules or may have been stabilized on an adhesion surface, the carrier can be a mixture 
20 of a solid suspension, micro-particles, embedded in a gel. Micro-particles may act not only 
as anchorage supports for injected cells, but also as anchorage supports for host-derived 
cells, for those particles not completely covered by previously seeded chondrogenic cells. 

This intended composition can be made in the form of an injectable combination of gel 
containing at least one hyaluronic acid derivative with a solid suspension containing at 

25 least an HA-derived particulate wherein the chondrogenic cells are evenly dispersed. Cells 
are harvested, isolated and expanded as described in Example 1 1 . Then, cells are mixed in 
a liquid medium containing at least one HA-derivative in form of fibres, granules, 
microspheres, nanospheres or sponge fragments made of an ACP or a benzyl ester 
derivative as described above. Cells are allowed to adhere to the micro-particles in a time 

30 comprises from 15 minutes up to 48 hours, or better 30 minutes up to 24 hours or more 

preferably 1 hour to 3 hours at room temperature, in the opiating room or even in a more 
controlled environment such as a cell culture incubator. Then, cells adhered to the micro- 



wo 00/37124 PCT/IB99/02077 

particles are embedded in the gel and, eventually, injected as described above. The 
combination of HA-derived components is such that a ratio between the micro-particle 
fraction and the gel fraction can be calculated. The optimal ratio is to be trimmed for the 
specific clinical application and may vary from 9 : 1 to 1 : 9 part of micro-particles over 
5 the gel. 

Example 12 - Treatment of bone non-unions with injectable combination of an HA- 
derivative carrier embedding a growth factor 

Bone non-unions are commonly complications occurring in orthopedic surgery when 
treating complex bone defects or dysmetabolism-affected patients. Actual state-of-the-art 

1 0 treatments for bone non-unions rely on drug administration or acellular biomaterial 

application. A relatively recent approach is to use a specific biological factors in order to 
stimulate host reparative system to overcome conditions that impede bone callus 
formation. Such biologically active molecules are, for instance, bone morphogenetic 
proteins (BMPs). However, since substances have to act locally and no to be dispersed by 

1 5 circulation system (vasculature and/or lymphatic), persons skilled in the art are testing 

various carriers. HA-derived compounds object of this invention are particularly suitable 
for this indication beause HA is not only osteo-conductive but also osteo-inductive. Thus, 
while releasing a certain amount of BMP, HA can also potentiate the effect of this 
biologically active protein and favour bone formation. 

20 The formulation to be used for bone ingrowth is either a gel embedding BMP, for instance 
BMP 2, or a combination of gel and micro-particles embedding BMP. Gel and micro- 
particles ratio can be trimmed as described above. This latter combination is mtended, but 
not limited to, to stimulate osteogenesis by direct HA action on bone precursors 
(osteoinduction), and also stimulate osteoconduction by tissue guidance. In addition, by 

25 combining, for instance, BMP 2 and a specific antibiotic, bone growth may be protected 
from infection, a common complication in bone non-unions. 

Example 13 - Treatment of cutaneous malformations bv injecting different cells in HA- 
derivatives based-gel and gel/solid combinations 

Cutaneous malformations have a significant impact on a peon's life quality, for instance 
30 after mastectomy or extensive bum injury of the face. State-of-the-art treatment protocols 
rely on the administration of tissue-augmentation degradable substances, for example 



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collagen. However, such temporary device do not eliminate permanently the anaesthetic 
character and need to be administered constantly. A stable augmentation can be achieved 
only if extracellular matrix is produced in a correct manner in order to re-establish the 
original skin contour. Cells injected in a liquid suspension are likely to be dispersed either 
5 by vascular or lymphatic system, thus loosing the capacity of synthesizing a permanent 
organized extracellular matrix. A carrier system, which guarantees a temporary stable 
anchorage to the surrounding tissue until a permanent adhesion occurs, can be constituted 
by an HA-derivative based-formulation. In addition, HA may, in part, acts directly in 
stimulating the wound healing process, as known in the literature. 

1 0 Thus, extra-cellular matrix-producing cells, such as fibroblasts, can be vehicled by 

embedding them in a HA-derivative based-gel as described above. Fibroblasts are then 
injected in subcutaneous space and sticked to the site until the natural process of adhesion 
to the surrounding tissues takes place. In alternative, a combination of micro-particles, on 
which first to attach fibroblasts embedded within a gel to deliver evenly dispersed cells, 

1 5 can be used. Ratio and compositions of different formulations are described above. Cells 
other than fibroblasts may be used to fill a cutaneous depression, such as, for instance, 
mammary glandular cells or adypocytes (either differentiated or uncommitted). 

Example 14 - Treatment of auto-immune diseases with genetic engineered cells embedded 
in injectable hvaluronate-derivatives solid/gel formulations 

20 Auto-immune diseases are due to a self-reactive response of the immune-system to specific 
body's factors, such as insulin in juvenile diabetes or cartilage tissue components in 
rheumatoid arthritis. Auto-immune diseases are chronic pathologies that affect million of 
people in the world. Current pharmacological protocols are focused on the 
symptomatology of the disease by giving generic anti-inflanmiatory substances delivered 

25 either locally or systemically with associated complications. New generation treatments 
will involve the use of more powerful and specific compound such as, for example, 
enzymatic inhibitors or receptor antagonists. However, permanent control of the diseases 
relies on the continuous administration of these substances with the risk of developing 
drug*related complications. Another forefi-ont solution is constituted, for instance, by the 

30 use of genetically transformed cells ex vivo to produce specific biological or 

pharmacological substances to counteract the immune reaction. In this particular 
application, cells are to be injected locally and they must maintain their viability for a long 



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PCT/1B99/02077 



time, possibly for the lifetime of the individual. For this purpose, cells must integrate in the 
application site, and this can be achieved giving a support in which cells are initially 
delivered and embedded. HA-derivatives described in previous examples can answer to 
this need, with the particular properties of being accepted in almost all compartment of the 
human body. Thus, as described before, chondrogenic cells can be harvested and isolated. 
Then, cells are transfected to express, in a bio-regulated fashion, anti-rheumatoid agents, 
such as, for instance, anti-IL-1 or anti-TNF-D, with techniques routinely used by persons 
skilled in the art, and expanded. Eventually, cells are delivered in rheumatoid arthritis 
patients in the same vehicle used previously. 

Another application of HA-derivatives based-formulations is constituted by the delivery of 
genetic material for in vivo gene therapy protocols. Using the combinations of HA 
described in example 10, DNA or RNA may be directly injected in a suitable carrier to 
transfect defective lung cells, such as those, for instance, involved in the cystic fibrosis 
disease. 

The invention being thus described, it is clear that these methods can be modified in 
various ways. Said modifications are not to be considered as divergences from the spirit 
and purposes of the invention and any modifications that would appear evident to an expert 
in the field come within the scope of the following claims: 



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CLAIMS 

1 . An injectable biocompatible composition comprising at least one hyaluronic acid 
derivative and at least one biologically or pharmacologically active component and/or 
mammalian cell, wherein said hyaluronic acid derivative is selected from the group 
consisting of: 

(a) a benzyl ester of hyaluronic acid wherein 50 to 75% of the carboxy groups are 
esterified with a benzyl radical; and 

(b) an auto-crosslinked derivative of hyaluronic acid wherein 3 to 15% of the carboxyl 
groups of hyaluronic acid are cross-linked to the hydroxyl group of the same or 
different hyaluronic acid molecule. 

2. The injectable biocompatible composition according to claim 1, wherein said benzyl 
ester is one wherein 50% of the carboxy groups are esterified with a benzyl radical. 

3. The injectable biocompatible composition according to claim 1, wherein said 
mammalian cell is selected from the group consisting of chondrocytes, osteocytes, 
fibroblasts, keratinocytes, adipocytes, muscle cells, nerve cells, cells from the 
peripheral nervous system, endothelial cells, hematopoietic cells, glandular cells, cells 
of the urethra and stem cells, both from aduk and embryo. 

4. The injectable biocompatible composition according to claim 1, wherein said cells are 
chondrocytes. 

5. The injectable biocompatible composition according to claim 1, wherein said 
biologically active component is a pharmacologically active component. 

6. The injectable biocompatible composition according to claim 5, wherein said 
pharmacologically active component is an antibiotic, an anti-inflammatory agent, an 
antiseptic, an active hormone, an anti-tumor agent or an anti-viral agent. 



10 



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7. The injectable biocompatible composition according to any one of claims 1 to 6, 
wherein said pharmacologically active component is a growth factor, or a 
differentiation/modulation faaon 

8. The injectable biocompatible composition according to claim 7, wherdn said growth 
factor is a member selected from the group consisting of transforming growth factor, 
insulin-like growth factor, platelet-derived growth factor, epidermal growth factor, acid 
and basic fibroblast growth factor, hepatocyte growth factor, keratinocyte growth 
factor, bone morphogenic proteins, and osteogenic proteins. 

9. The injectable biocompatible composition according to claim 5, wherein said 
pharmacologically active component is a nucleic acid such as DNA and RNA. 



10. The injectable biocompatible composition according to anyone of the previous claims 
1 5 which further comprises fibres, granules, microspheres, nanospheres or sponge 

fragments of a derivative of hyaluronic acid. 

11. The injectable biocompatible composition according to claim 10, wherein said 
derivative of hyaluronic acid is the total benzyl ester derivative. 

20 

12. Use of at least one derivative of hyaluronic acid for the injection administration of a 
biologically or pharmacologically active component or cell, wherein said hyaluronic 
acid derivative is selected from the group consisting of 

25 (a) a benzyl ester of hyaluronic acid wherein 50 to 75% of the carboxy groups are 

esterified with a benzyl radical; and 
(b) an auto-crosslinked derivative of hyaluronic acid wherein 3 to 15% of the carboxyl 

groups of hyaluronic acid are cross-linked to the hydroxy 1 group of the same or 

different hyaluronic acid molecule. 
30 13. Use according to claim 1 2, wherein said benzyl ester is one wherein 50% of the 

carboxy groups are esterified with a benzyl radical. 



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14. Use according to claim 12, wherein said mammalian cell is selected from the group 
consisting of chondrocytes, osteocytes, fibroblasts, keratinocytes, adipocytes, muscle 
cells, nerve cells, cells from the peripheral nervous system, endothelial cells, 
hematopoietic cells, glandular cells, cells of the urethra, stem cells and genetically 
modified cells, both from adult and embryo. 

15. Use according to claim 14, wherein said cells are chondrocytes. 

16. Use of an injectable composition for repair of cartilage, wherein said composition 
comprises chondrocytes in combination with a hyaluronic acid derivative selected from 
the group consisting of 

(a) a benzyl ester of hyaluronic acid wherein 50 to 75% of the carboxy groups are 
esterified with a benzyl radical; and 

(b) an auto-crosslinked derivative of hyaluronic acid wherein 3 to 15% of the carboxyl 
groups of hyaluronic acid are cross-linked to the hydroxyl group of the same or 
different hyaluronic acid molecule. 

17. Use according to claim 16, wherein said benzyl ester is one wherein 50% of the 
carboxy groups are esterified with a benzyl radical. 

18. Use of at least one derivative of hyaluronic acid for the protection during storage and 
transportation of a mammalian cell, wherein said hyaluronic acid derivative is selected 
from the group consisting of 

(a) a benzyl ester of hyaluronic acid wherein 50 to 75% of the carboxy groups are 
esterified with a benzyl radical; and 

(b) an auto-crosslinked derivative of hyaluronic acid wherein 3 to 15% of the carboxyl 
groups of hyaluronic acid are cro^s-linked to the hydroxyl group of the same or 
different hyaluronic acid molecule. 

19. Use according to claim 1 8, wherein said benzyl ester is one wherein 50% of the 
carboxy groups are esterified with a benzyl radical. 

20. Use according to claim 1 8 or 19, wherein said mammalian cell is selected from the 
group consisting of chondrocytes, osteocytes, fibroblasts, keratinocytes, adipocytes, 
muscle cells, nerve cells, cells from the peripheral nervous system, endothelial cells. 



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hematopoietic cells, glandular cells, cells of the urethra, stem cells and genetically 
modified cells, both from adult and embryo. 

21 . Use according to claim 20, wherein said cells are chondrocytes. 

22. A method for the treatment of cartilage damage which comprises injecting into the 
intra-articular space of a patient a composition which comprises chondrocytes in 
combination with a hyluronic acid derivative selected from the group consisting of 

(a) a benzyl ester of hyaluronic acid wherein 50 to 75% of the carboxy groups are 
esterified with a benzyl radical, and 

(b) an auto-crosslinked derivative of hyaluronic acid wherein 3 to 15% of the carboxy! 
groups of hyaluronic acid are cross-linked to the hydroxyl group of the same or 
diflTerent hyaluronic acid molecule. 

23. A method according to claim 22, wherein said benzyl ester is one wherein 50% of the 
carboxy groups are esterified with a benzyl radical. 



INTERNATIONAL SEARCH REPORT 


Ml Jonal Apfiilcallofi No 

PCT/IB 99/02077 


A. CLASSmCATTON OF SUBJECT MATTER 

IPC 7 A61L27/38 




AoooRfim to Irrtemattonal Paiont daasWcatton (IPC) or to bo«h national daseificatton and IPC 




a FIELDS SEARCHED 


Mlr*iuim documamation aeaiched (claaeilication ayatem followad by ciaaeification aymboto) 

IPC 7 A61L A61K 



[Xwumantatkjnaoarohad other than mhimum docunwntation to the axtem that auch documanta ara Included in the fiekto aeaiched 



Bactronlc data baaeconautted during the htematlonal aearch <name of data Iwae and, where piarticel, •aarchtaimauaad) 



CMgoiy* 


Clallan o< daeuRMnt, witti MeMion. when apprapriale, ol0ie ralatrara paaugaa 


Relevant to Claim No. 


X 


WO 97 45532 A (UNIV BROUN RES FOUND) 

4 December 1997 (1997-12-04) 

page 5, line 26 -page 6, line 25 

page 7, line 18 - line 30 

page 9, line 9 - line 24 

page 11, line 31 -page 13, line 25 


1,3-12, 
14-16,22 


X 


UO 98 56897 A (ABATANGELO GIOVANNI 
;CALLEGARO LANFRANCO (IT); FIDIA ADVANCED 
BIOP) 17 December 1998 (1998-12-17) 
claims 


1-3,7,8, 

10-14, 

22,23 


X 


UO 97 49412 A (CALLEGARO LANFRANCO 
;PAPARELLA ANNAHARIA (IT); BELINI DAVIDE 
(IT);) 31 December 1997 (1997-12-31) 
claims 


1.5-8, 
12,22 



m 



Further docuntenta are Uated in the continuation of box C. 



Paterti family membera am Qated In annex. 



* Special categortoa of dted doctanema : 

•A' document defining the ganaralatate of the art which la not 

oonaktofed to be of particular relevanoe 
•E' aaiiiar document but pubUahed on or after the intemaHonal 

filing data 

T' document wMch may throw doubto on priority ctaim(a) or 
which is dtad to eetabllBh the publication data of another 
citation or otharapecia)reaaon(aa apeclfied) 

Ky document mfening to an oral dadoeure.uae. exhibition or 
other meana 

'P* dooumer«pub6ahed prior to the htametionat fBingdatebut 



T later dooumertpubllahad after the tntemationat filng date 
orprtorttydateandnotinconffiotwththe application but 
cited to underatand the principle or theory undartylngthe 
invention 

•X" document of particular relevanoa: the dalmad invention 
cannot be conaidared novel or cannot be oonaidated to 
tnvdwaninvarithwaiapwhenthedocumeritla taken alone 

"Y* document of particular lelevanoe; the claimed invention 
cannot be oonaidated to tnvotve an inventive atepwhenthe 
document ta combined with one or more other auch docu- 
ment*, euch combination being obvioua to a paraon aidHed 
tnthaart 

*&* document member of the aame patent family 



Date of the actual completion of the International aearch 

17 April 2000 


Date of mailing of the international aearch report 

02/05/2000 


Name and mallng addreaa of the ISA 

European Patent Office. P3. 5618 Patandaan 2 
NL-2280HVRI|nvl9t 
Tel. (431-70) 340-2040. Tx. 31 6S1 apo nl. 
Fax; (431-70) 340-3016 


Authorized officer 

Thornton, S 



FomPCT/tSAaiO(( 



M)gu|yi902) 



page 1 of 2 



INTERNATIONAL SEARCH REPORT ii<».iAi>pncMonNo 

PCT/IB 99/02077 


C^ConltnuMlan) OOCUMEMTS CONSIDERED TO BE RELEVAHT 


Catagny ' 


Cttatton of document, with frKication.wh«r» appropriate, of the retevant paaaagoa 


Retovanto daim No. 


P,X 


UO 99 24070 A (CALLEGARO LANFRANCO ;RENIER 
DAVIDE (IT); FIDIA ADVANCED BIOPOLYHER) 
20 Hay 1999 (1999-05-20) 
page 7, line 6 - line 27 
claims 


I- 8, 

II- 23 



page 2 of 2 



INTERNATIONAL SEARCH REPORT 

tefoiintUoft on ^•nt timlly n>an*e« 


littv -JonalAppllesllonNo 

PCT/IB 99/02077 


Patent document 
cited in search leport 


Publication 
date 


Patent tamSy 
menrtberte) 


Publication 
dote 



UO 9745532 



04-12-1997 



US 5939323 A 



17-08-1999 



NU ffOwwo;?/ 


A 


17-12- 


-1998 


IT 


PD970122 A 


11-12-1998 








AU 


8536898 A 


30-12-1998 










EP 


0985029 A 


15-03-2000 


wo 9749412 


A 


31-12- 


■1997 


IT 


PD960163 A 


22-12-1997 








AU 


3435497 A 


14-01-1998 










CA 


2258920 A 


31-12-1997 










EP 


0954323 A 


10-11-1999 


UO 9924070 


A 


20-05- 


■1999 


IT 


PD970253 A 


06-05-1999 








AU 


1559899 A 


31-05-1999 



form PCTASAiSlO (pB»al Un*f mt>%x) Wu(y 1862) 



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