Title of Invention


Abstract A dihydrophenyl XXX cross-linked macromolecular network is provided that bis in articles XXX and XXX and XXX application such as a XXX synthesis catridge.The ntework is made by first providing a polyamines or polycarbomate myclonucleotide xxx a secquirity of amine or carbonyle and respectively structured along the length of the molecule.re acting thus macroXXX with a XXX compound having a free carboxylic acid group in the case of a polycrane or a XXX primary smine group molecule of a polycarbonate and subsdrung the hydroxyphenyl compound onto the macromelecule via ii embodiment obtained XXX pathway to provide a hydroxyphenyl substituned macromolecules.This macromolecule is then linked to other such macro XXX an enzyme catalysed dimenzatoic reaction between two hydroxy groups attached respectively in different mesurement under XXX condition of tempareture and pH.In a prefered embodiment the thereto molecules network is made up hydramine substutents XXX molecule that are visited by dimethyl loads to provide a stable cohherent hydrogen with detail physical propetides.A method of preparing such a network is also provided.
Full Text function in healthy joints, ft is responsible for absorbing and dissipating impact and factional bads in order to divert these loads away from bones, to protect t' ie bones ftom damage. Cartilage perfonns this function by transferring the loading force to a flnid phase within a thKse-diinensional network of aggrecan molecules, themselves constrained (described in the next ]3aragraph) within the joint space. Aggrecan molecules ha re up to 100 chondroitm sulfate dtains attached to a cote protein, with each chondroitin sultatit chain possessing multiple negatively charged sulfate groups along their length. The efleel of all these sulfate groups is to cause each of the cbondroitin sulfate chains in a single aggrecan molecule to repel one another, (resulting in the aggrecan molecule having the maximum possible volume at rest), and also tc cause adjacent aggrecan molecules in a cartilage aggregate to repel one another.
In healthy cartilage, aggrecan molecules are attached to long hyaluronan chains, which are in rum coast rained in large cartilage aggregate!! within the joint space by an extracellular collagen fibril matrix Thus, even though acjacent chondroitin sulfate chains in each aggrecan mob:til; (and adjacent aggrecan molecules attached to the same or a different hyaluronan chain) repel one another, they are nonetheless, constrained within the collagen matrix. See Fig. 1 depicting normal, healthy cartilage. Because the chondroitin sulfate chains are so repulsive, the hyaluronan-aggrecan network (or macroinolecular network) expands as much as possible within the constraints of the collagen matrix to achieve the lowest possible energy state at rest; Le. to allow the maximum possible spacing between adjacent negatively ckirged sulfate groups. As a result, network molecules are highly resistant to being shift
WO 2004/063388 PCT/L'!J2004/000478
cartilage and its ability to resist deformation and absorb coinpressive loads, further described below.
Within the nwcn >xnolecular network are water molecules which provide a substantially continuous fluid phases The macromolecular network diverts impact and ftictional loads away fix m bones by transferring them to the continuous fluid (water) phase as follows. As a joint undergoes a load, the force is absorbed first by the macromolecular network, where it acts o a and tends to deform or compress the network. The force sets up pressure gradients in th Through thia elegant mechanism, normal cartuagu is capable of absorbing significant loads by transferring the bulk of the loading force to a fliid phase constrained within a macromolecular network. This arrangement has yet to bs adequately duplicated via artificial or synthetic means in 1 he prior art Consequently, there is no adequate remedy for cartilage degenerative disorders, such as arthritic disorders, where the aggrecan molecules become separated from their h /aluronan chains and are digested or otherwise carried out from the cartilage aggregates.
Osteoarthritis iind rheumatoid arthritis affect an estimated 20.7 and 2.1 million Americans, respective ly. Osteoarthritis alone is responsible for roughly 7 million physician visits a year. For severe disabling arthritis, current treatment involves total joint replacement with on average 168,0 00 total hip replacements and 267.000 total knee replacements performed per year in the U.S. alone. Defects in articulsr cartilage present a complicated treatment problem because of the limited capacity of chemdrocytes to repair cartilage. Treatment strategies t) date have focused on the use of {intologous chondrocytes expanded in

WO 2004/063388 PCT/US2004/00047S
culture or the recrukraw it of tnesenchymal stem cells in viw by chemotactic or mitogenic agents. The intent oi thse strategies is to increase and/or activate the chondrocyte population so as to resy ithesi?e a normal, healthy articular cartilage surface. One major difficulty associated wiih these strategies is the inability to maintain these agents at the site of the defect Byalurocan has been proposed as a candidate for the development of biomaterials for local delivery of chondrocytes or btoactive agents because of its unique properties, including ex ceUent Incompatibility, degradability, and theological and physiochemical proper! ies. However, it has been unknown whether chondrocytes suspended in a tissue engineered t yaluronan matrix would be able to synthesize a new cartilage matrix with mechanical prop© ties comparable to normal, healthy articular cartilage. This is because conventional bioma!:eri als made from hyaluronan are formed through chemistries that are incompatible with mail itaiaing cell viability. Chondrocyi es must be introduced to the matrices after matrix fi irmation with variable and normally poor results.
Accordingly, tl .ere is a need in the art for an artificial or synthetic matrix that can effectively divert a loa ling force from bones in an effective manner. Preferably, such a matrix can be proviiecI in situ or in vivo to repair or repk&e articular cartilage during an orthopedic surgical procedure. Most preferably, the artificial or synthetic uiatrix can be provided to an in situ r in vivo target site as a liquid or a. plurality of liquids, and can set up in place to provide a si ibstantially seamless integration with existing cartilaginous and/or bony tissue in a patien t.
SUMMARY OF THE INVENTION A macromolec alar network is provided including the following structure

wherein Ri am 1R2 each is or includes a structure selected from the group consisting of polycarboxylates, rolyamines, poryhydroxvpheayl molecules, and copolymers thereof, and wherein Ri and Rz can be the same or different structures,
A macromolet ular network is also provided having a plurality of tvraaiine-substituted hyaluronan molecules, wherein at least two adjacent hydurooan molecules are finked by a dhyramine linkage.

WO 20(14/063388 PCTWS2004/000478
A hydrogel is sis > provided which includes a macromolecular network of tyramine-substituted hyaluronaa molecules that are cross-linked by clityramine linkages between hyalurcman molecule}!.
A method of ical iog a macromolecular network is also provided including the steps of providing a first maaoniolecular species selected from the group consisting of hydroxyphenyl-substitu :ed porycarboxylates, hydroxypheiiyl-substitutedpolyamines, other polyhydroxyphenyl molecules, and copolymers thereof, and forming at least one dihydroxyphenyl linkag e between two hydroxyphenyl groins attached respectively to adjacent ones of the first macromolecular species.
A method of nuking a hydrogel is also provided having the following steps: a) providing a first solu lion having other a peroxidase enssyme or a peroxide but not both, and also a macromoxxilar species selected from the group consisting of hydroxyphenyl-substituted polycarboxylaies, hydroxyphenyl-substituted polyamines, other polyhydroxyphenyl molecules, and copolymers thereof; b) providing a second solution having the one of the peroxidase enzyme or peroxide not provided in the first solution; and c) combining the first ind second solutions to initiate dihydroxyphenyl cross-Unking to form the bydrogel.
Fig. 1 is a schematic diagram of normal, healthy human cartilage.
Pig. 2 is a schematic diagram of a dihydroxypheaayl cross-linked macromolecular network according to Fig. 3 is a stnxiural formula of a hyaluronan molecule.
Fig. 4 is a gxap h showing comparative results for mechanical testing in a confined compression test (equilibrium stress versus applied strain) of T-HA hydrogels according to the invention versus p iblished results for articular cartibige plugs (Example 3).
Fig. 5 is a graph showing comparative data of glucose utilization for chondrocytes embedded in T-HA hj drogels (1.7% and 4.7% T-HA) compared to cultured on tissue culture plastic (control).
As used hereir, the term polycarboxylate means a molecule, structure or species having a chain lenjjth of at least two functional groups or units, wherein at Jeast two such

WO 2004/063388 PCTXS2004/000478
groups or units of the ehiin are or comprise caiboxylic acid groups that arc sterically accessible to a nuclecph ilic substitution reaction as described herein. Also as used herein, the term polyamine means i molecule, structure or species having a chain length of at. kast two tunctioaal groups or jd is, wherein at least two such groups or units of the chain are or comprise primary airin<: groups that are available for a nudeophih substitution reaction. also as used herein p bryhydroxyphenyl molecule mean.1 having chaw length of at least two function or units wherein such the chain comprise h ydroxyphenyl can be linked to anotbet hydroxyphenyl group via c-c bond hydrogel is material prepared comprising macro mo scalar network usdiul in tissue replacement engineering applicatiois e.g. artificial cartilage coat surgical instruments prevent irritati on provide semi-permeabk membrane use an kidney etc.> The invention i icludes a novel structure of a maaomolecular network that has been formed by linking hydi oxypheiiyl groups attached to adjscent long chain macronolecules, resulting in effectively cross-Unking the macromolecules to provide a large network. The basic cross-Unking stricture of the network is shown below

where Ri and R2 are a tch long chain macromolecules. B4 and Ra can be the same molecule or different molecules: but it will be understood tiiat to provide a suitable network, Ri and Ra will be different mole By providing 1 plurality of these dihydroxyphenyl linkages between adjacent macromolecules, a nei work of dibydroxyphenyl cross-linked macromolecules is provided as shown schematically in Fig. 2. In the figure, the macromolecules are represented schematically by cylii drical strands, each having at least two hydroxyphenyl groups attached along ha length. It is: loted that not every hydroxyphenyl group must be linked to another hydroxyphenyl group

WO 2004/063388 PCT/US2004/000478
Briefly, the disclosed invention involves covalent coupling of hydroxyphenyl containing compounds, i Deluding but not limited to tyramme, through their primary amine (or carboxyl) groups to carl oxyi (of primary amine) groups on various polynxeric scaffold materials, including but cot limited to hyaluronan or chondroitin sulfate (e.g. in the form of aggrecaa). via a carbodi imide-inediated reaction. After isolation and purification of the hydroxyphenyl-substituied polymeric scaffolds, the hydratyphenyl residues are selectively cross-linked by horsarailish peroxidase (HRP) in the presence of very dilute hydrogen peroxide to form hyrilroi;els.
The first step in providing the macromolecular network is to prepare or provide the long-chain macromolec ales having periodic hydroxyphenyl groups attached In one embodiment, the macic molecules are poiyhydroxyphsnyl molecules which already have multiple or periodic hy iroxyphenyl groups, such as polyphenols. Suitable polypheaols include polyamino adc3 (e.g. polytyrosine), epigallocatedua (EGC), and epigallocatechin gallate (EGCG) isolate i from green tea, less preferably oiier poryphenols.
In a further emludiment, the hydroxyphenyl groups can be added to the macxomolecules perioc iically or randomly along their length via a chemical reaction. A preferred method of ad ding hydroxyphenyl groups to the macxomolccules is to utilize a carbodiimide-medktec substitution reaction pathway to provide an amide bond between a primary amine having i hydroxyphenyl group and a carboxylic acid group attached to the macromolecules. Jbt tl is method, the long-chain macromolecule prefierably is a polycarboxyiate mo lee ule, havmg periodic carboxylic acid groups along its length. The hydroxypbenyl groups are provided as part of smaller molecules having primary amine groups that can be atta ched to the carboxyl carbon atoms of a carboxylic acid group on the long-chain macromol* cules via the carbodumide pathway. The reaction proceeds as follows:

WO 2004/06338S PCT/US2004/000478

Structure A is a carbodiimide;
Structure B is J polycarboxylate (though only on« COjH group is shown);
Structure C is the product of Reaction A and is ait activated O-acylisourea;
Structure D is; i primary amine having a hydroxyphenyl group;
Structure £ is s. hydroxyphenyl-substrtutedpolycffboxyJate; and
Structure F is i n acylurea byproduct;
wherein individual Rs can be individually selected, the same or different from one another, to be a stndg] it chain or branched alkane or acy) group, ox any other stmcture that docs not interforc v/M. the carbodiunide reaction pathway to provide the amide bond between the NH2 and CO2H gr jups as shown in Structure E above.
In the above-i] lustrated pathway, Reaction A repnesents a carbodiimide activation of the carboxyl group to provide an activated O-acyUsoureii intermediate. Hie electropositive carbon atom of tbi'i va termediate is receptive to nucleopldlic attack b>r the lone pair of electrons on a nitrcgei atom of an adjacent primary amke molecule having an attached

WO 2004/0C3M8 PCTAJS2004'000478
nydroxyphenyl group. The products of this nucleophilic substitution reaction (Reaction B) are a hydroxyphenyl-s ibstituted polycarboxylate and an ;jcylurea byproduct which can be dialyzed out to provide a substantially pure hydroxyphenyl-substitute polycarboxylate product,"
Certain side-re actions are possible in the above-described carbodiimide reaction pathway chemistry an 1 should be considered by the person having ordinary skill in the art. First, the carbodiimid*: can react withnucleophiles other than the carboxylate oxygen atom of the polycarboxylate nrolecule required to form the desired O-acylisourea (reaction A). Such nucleophiles may include the amine and/or hydroxyphenyl groups of Structure D illustrated above. In particular, 1 here are three potential side-reactions for Reaction A which can reduce the effective conceotr. rtion of the carbodnmide and the primary amine having the hydroxyphenyl group (Structures A and D), and potentially lead to the creation of undesired adducts on the polyca rboxylate (Structure B):

WO 2004/063388 PCT/US20O4/000478
The product of an amine reaction with the carbodiimide (Reaction C) will not have a free amine group effectively reducing the amount of tyramine available for reaction with the O-acytisourea. This reaca Once the desire i O-acyiisourca product has been formed in Reaction A, there is again the possibility for certam additional side-reactions:

WO !004/t)63388 PCT/US2004/000478

The O-acyliso jrea (Structure C) can be hydrolyzed as shown in Reaction F releasing the original uumodiiu d polycarboxykte (Structure B) and the ecyturea of the carbodiimide (Structure F). This is an unproductive reaction similar to reaction E, which reduces the effective concentratioa of the carbodiimide. The O-acylisonrea, can also undergo an intramolecular rcatrai igement (Reaction 0) to fbon two unreactive N-acyliseas. These structures form unproductive adducts on the carboxylatc: molecule which cannot contribute to the peroxidase catalysed cross-linking reaction shown (sitep 2 discussed below) for preparing

WO 2004/063388 PCTAJS2004/000478
the network according to the invention. The O-acylisourea can also react (Reaction H) with a second carboxyl group 0:1 either the same or a different polycarboxylate molecule to form an acid anhydride. This mo tecule can then react with Structure D to form the desired amide and regenerate the second carboxyl group. Thus there are two potential side-reactions for the 0-acylisourea, which can r sduce the effective concentration of the carbodiimide (Reactions F and G), and potentially 1 ?ad to creation of undesired addncts on the polycarboxylate molecule.
Negative effects of these side reactions can be addressed through conventional techniques without undue experimentation.
Alternatively to the pathway shown above where the macromolecule (Structure B) is a polycarboxylate, the nucroniolecule can be a polyamine having multiple or periodic amine groups along its length, wherein the hydroxyphenyl groups then are provided as part of smaller carboxylic acid molecules. Suitable polyamines include: pofyhexosamines such as cbitasan (polyglucocanine); polyamino adds such as polylysine; polydeoxyribonuclcotides such as poly (dA) (polj deoxyadenylic acid), poly(dC) (pcilydeoxycytidyHc acid), and poly(dG) (polydeoxygiianylic acid); and polyribonucleotides such as poly(A) (polyadenylic acid), poly(C) (polycytidylic acid), andpoly(G) (polyguaaylic acid). The carbodnmide-mediated reaction pathway proceeds exactly as explained above to form the aioide bond between the amine &o up and carboxylic acid group except that, as will be understood by a person having ordinal} skill in the art, the resulting product will be hydroxyphenyl-substkuted polyamiae instead of a polycarboxylate. Other peptides and/or proteins also can be used as the macromoleculcs in the present invention, either which have hydroxyphenyl groups disposed along their length, or to which hydroxyphenyl groups can he provided via a substmition reaction a: described herein. For example, in addition to the peptides already . disclosed herein, poly: xginine can be used as the macromolecule.
When substituling onto a polycarboxylate molecule, suitable hydroxyphrayl* containing compound1 for use in the present invention include those having a free primary amine that can be used to modify scaffold materials having multiple or periodic CO2H groups, including tyro sine (2-amino-3-(4-hydroxypbenyi) proprionic acid) andtyramine (tyrosamine or 2-(4-h;»droxyphenyl) ethylamine). When substituting onto a poryamine, suitable hydroxypbenyl-containing compounds include those having a free COzH group that can be used to modify scaffold materials having multiple or periodic primary NH2 groups, including tyrosine, 3H4-bydroxyphenyl) propwnic acid jffld 4-hydroxypheoylacetic acid.

WO 2004/063388 PCT/US2004/000478
The second step. n preparing the cross-linked macromolecular network according to the invention, is to lias tJ is resulting macroinolecules, now having one ox more hydroxyphenyl groups attached, via EL di hydroxyphenyl linking structure. In this step hydroxyphenyl groups attached to different maaromolecules are linked via the reaction mechanism skwn below using a peroxide reagen: in the presence of aperoxidase:

WO 2004/063388 PCT/US2004/000478

WO 2004/063388 PCTT.TW004/000478
(it is noted that ^ me dihydroxypheuyl linking may occur between different hydroxyphenyl groups 011 the same molecule as well). Peroxidase in the presence of a dilute pea-oxide (preferably H2C >2) is able to extract the phenolic hydroxyl hydrogen atom from hydroxyphenyl containirg compounds (such as tyramine) leaving the phenolic hydroxyl oxygen with a single unj bared electron, an extremely reactive free radical. The free radical isomerizes to one of the two equivalent ortho-position carbons and then two such structures dimerize to form a ceva ent bond effectively cross-linking lie structures, which after enolizing generates a diiydroxyphenyl dimer (a dihydroxyphenyl linkage such as dityramine linkage as described below).
For clarity, o:aly a single dihydroxyphenyl Unking reaction is shown above, but it will be understood that swe ral or multiple such linkages will he produced when macromolecules having attached hydrox yphenyl groups are subjected to the reaction conditions (peroxide and peroxidase). Hydrogen peroxide is indicated in the above marhMiism, but other suitable peroxides can be used. Also, the peroxidase preferably is horseradish peroxidase (HRP). Alternatively, any oiiie - suitable enzyme (or other agent) can be used that is capable of generating free-radical: 1 for cross-Unking scaffold materials containing hydroxyphenyl groups, preferably uodjr ordinary metabolic conditions as described below.
The dihydioxy] ihenyl cross-linked macromolecultir network is superior to conventional cartilage it other tissue replacement or substitution methods and products because the cross-linking reaction is enzyme driven (peroxidase). This means the cross-linking reaction is carried out under ordinary in vivo or metabolic conditions of temperature such as 35-39°C (e.g. about 37°C), pH range of 6-7 (e.g. about 6.5), reagent* etc. (A peroxide, such as hydngen peroxide, is the only required reagent for the cross-linking reaction). Thus, th« a oss-linking reaction can be performed in vivo, to provide a cross-linked hydrogel at a surgical situs, such as an orthopedic surgical situs, to promote maximum seamless integration b stween the hydrogel and native tissue such as bony and cartilaginous tissue. Integration of 1 he new hydrogel scaffold with native cartilage matrix may occur immediately as the hy iroxyphenyl-substituted macromolecular scaffold quickly penetrates into the existing cartil;ige matrix prior to cross-linking, and cross-links not only with other hydroxyphenyl-substi uted macromo Jecular scaffold material but potentially with tyrosine residues of resident pi oteins in the existing cartilage matrix. This would eliminate a typical problem found with p -©-formed matrix plugs, which is their poor integration, into the native cartilage tissue. The
WO 2004/06338$ PCI7US20W7000478
eliminates the need to s' jrgically enlarge a defect to fit a pre-cast plug, as is necessary for hydrogels whose chemi rtries are toxic to or otherwise proiiibit their formation inside the patient. It should be no ted that most cartilage damage as n result of arthritis presents as a variable thinning of ihe articular surface, not holes of defined shape.
Because the cro js-linking reaction requires both tie peroxide and a peroxidase (preferably horseradish peroxidase), solutions containing nil but one of these components can be prepared for com en ieot application to a surgical site. For example, a solution comprising a tyramine - (or otter bydtoxyphenyl containing species) substituted polycarboxylate (such as tyramine-substitutecl h; 'aluronan, etc.) and the peroxidase can be prepared, with a second solution prepared conti lining the peroxide. Alternatively, the peroxide and the peroxidase can be swapped between tie first and second solutions, the important thing being that the peroxide and peroxida: ;c are kept separate (Le. hi separate solutions) until the cross-linking reaction is to be carriei I out. Then, the first solution is applied, (e.g. to an in vivo surgical situs), and the second: lolution is applied or sprayed over the first, in vivo, to cause in situ cross-unking of the tyi amine residues. The cross linking reaction occurs in vivo. Other combinations will be e vident from the present disclosure which are within the skill of a person of ordinary ski] 1 in the art.
Furthermore, because the cross-linking reaction cccurs under ordinary metabolic conditions, additional living cells, such as choodrocytes, progenitor cells, stem cells, etc., can be provided directly tc a medium containing the non-cro js-linked hydroxyphenyl-substituted polycarboxylates or polyamines (or polyphenob), i.e. to the first or second solution from the preceding paragraph, therein the cell-rich medium is applied with the inacromolecules to the site in vivo, and the m) Jecules are subsequently cross-linked via addition of peroxidase and peroxide. The result i 5 a cross-linked macroxnolecular artwork containing the desired cells dispersed within it. S In a preferred zmbodiiuent particularly suitable for preparing synthetic curtilage as well as other synth«i
WO 2004/063388 PCT/US2004/000478
according to the invcrttio a is hyaiuronan 01 hyaluronic acid (HA), and the hydroxyphenyi group is supplied in the ibnn of tyramine.
HA is composed of repeating pairs of gbcuronic acid (glcA) and N-acetylglucosamine (gl
Structure A is EDC;
Structure B is ayaluroEan (though only one CQ2H group is shown);

WO 2004/063388 PCT/US2U04/000478
Structure C is the product of Reaction A and is l-e1hyl-3-(3-dimethylaB5inopropyl)
Structure D is tyramine;
Structure E is ty ^mine-substituted hyaluronan; ami Structure F is 1 - sthyl-3-(3-dimetbylaininopropyl) urea (EDU). In the above pat away, a negatively charged oxygen atom of the carboxyl group of the hyaluronan molecute el lacks, via a nucleophilic reaction mechanism, the electron-deficient diimide carbon atom or; the carbodiimide molecule (EDC) to form the activated O-acylisourea (Reaction J 0- The result 13 that the carbon atom of the HA carboxylate group becomes sufficiently electron deficient to be susceptible to nucleophilic attack by the unshared pair of electrons on the amine group of a tyramioe molecule (Reaction B). Reaction A is preferabl y catalyzed by a suitable catalyst that will result in the formation of an active ester daring Rea ction A, thus permitting the reaction to be carried out at substantially neutral pH (e.g. pH»6.5). Suitable catalysts include N-h)droxysuccinimide (NHS), less preferably l-hydroxybjnzotiiazole (HOBt) or N-hydrox>sulfosuccinimide (NHSS), less preferably another suitable catalyst or combinations thereof effective to enhance the carbodiimide reaction by formation of an active ester in order to minimize the unproductive hydrolysis of carboidin aides at higher pHs. Less preferably other carbodiimides besides EDC can be used, including l The result of Reaction A above is 0-acylisourea-;nibstituted hyaluronan; essentially the EDC molecule has been temporarily substituted onto the carboxylic acid group of a glcA residue from the HA t jolecule, making the carbon atom of the carboxylic acid group slightly positively charged. T! ie electron pair from the terminal umine group of a tyramine molecule is then substituted ont > the carbon atom via a nucleophilic substitution reaction as explained in the preceding paragraph (Reaction B). The result of BLeaction B is the tyramine-substituted HA molecule (T-HA) and acylurea, a byproduct. It will be understood mat Reactions A and. B will result in a plun ility of tyramine substitutions on ttie periodic glcA residues of HA molecules; a single substitution has been shown here for brevity and clarity.
After formation of T-HA, a plurality of T-HA molecules are reacted via peroxide and peroxidase enzyme to cross-link T-HA molecules as previously described and illustrated above. That is, the hj droxyphenyl groups on. the tyramine residues now attached to HA molecules react with peroxide (preferably H2Oz) in the presence of a peroxidase to remove

WO 200 18
the phenolic hydrogen ai oro resulting in a tyramine free radical, with the unpaired electron associated with the phenolic oxygen atom. This free radiwil species isomerizes or resonates, resulting in a resonance structure (or free radical isomer) with the unpaired electron now associated with an ortho carbon atom on the phenolic ring, In this position, the unpaired electron quickly reacts i rith a similarly situated unpaired electron on another tyramine free radical to form a covale at bond therebetween. The result is a free-radical driven diinerization reaction between difiHen mt tyramine free radical residues attached to differec; glcAs of ihe same or different HA n olecutes. This dimerized species 1'urther enolizes to restore the now-linked tyramine residue s, resulting in a dityramine linkage structure. It will be understood that a plurality of reactions as herein described will occur between adjacent tyramine residues, resulting lit a z-oss-linked macromolecular network of T-HA molecules according to the invention having the following cross-linking structure:

The cross-linked T-HA network can be provided with aggrecan molecules in a conventional manner, e.g. via link proteins, to provide a cross-naked T-HA network having aggrecan molecules attached to the HA chains. Thus, a network similar to that found in a normal cartilage aggregate cai be provided according to the invention, with the dityramine bonds holding the network together thereby constraining the contained aggrecan network, instead of collagen fibrils as in cormal cartilage.
It will be understood from the present invention ihat other glycosaminoglycans, polysaccharides and polycarboxylic acids can be used as; the macromolecules:for producing the cross-linked netw vk disclosed herein. For example:, suitable glycosaminoglycaos, other than HA, include choiidroitin, chondroitin sulfate, dennutan sulfate, heparan sulfate and heparin. Other suitab Is polycarboxylates include: proteoglycans such as versican, aggrecan, and cartilage aggrega«s composed of aggrecan, hyahironan and hnk protein; polyuronic

WO 2004/06338S PCT/US2004/000478
acids suck as polypeci:ai<: acid polyglucuronic pectin a: ethyl ester colominic and alginate amina acids at least amino units that meet the definition of polycarboxylate given above such as polyaspartic polyglutamic ml these can be substituted with one or a plurality hydroxyphenyl groups i sing carbodiimide-mediated reaction pathway disclosed herein by person ordinary sldi in art without undue experimentation.> As mentioned al >ove, it is also to be understood that native polyphenol compounds, which already contain t vo or more hydroxyphenyl groups that can be cross-linked using the described enzyme catal /sis chemistry can be used in place of the polycarboxylates and polyamjnes described a bove which must have the hydrox^hesyl groups added by a chemical reaction.
In another piefc rred embodiment, a network of tyi amine cross-linked chondroitin sulfate molecules (e:ilhulsive force contributing to the compression resistance of the network aggregate while the tyramine crosslinks constrain the chc ndroitin sulfate network from breaking or dissipating. Tbs result is a similarly non-dispLiceable chondroitm sulfate network (nod concomitant water-impermeability) as in normal cartilage, but without the extracellular collagen fibril matrix or the HA chains found i a normal cartilage. In fact, by dir
WO 2004/063388 PCT/US2004/000478
interstitial fluid phase is even more constrained from fiowiag, In this embodoneBt, where chondroitin sulfate molecules are directly cross-linked, certain cartilage degenerative conditions are entirely cicumvented; e.g, conditions where the core protein to which chondroitb. sulfate raoit cules are ordinarily bonded in normal cartilage becomes cleaved between the HA binding; domain (Gl) and the second globular domain (02) thus allowing the cbondrottin sulfate rich region to diffuse out from the cartilage aggregate. In this embodiment, because tl le chondroitm sulfate molecules are directly cross-baked to one another, unassociatdi \» ith an aggrecan or otlier proteoglycan molecule, they cannot be cleaved or carried away as in normal cartilage.
Nonetheless, a 1 yramine cross-linked T-HA network (having an HA backbone chain with attached aggreion molecules, which in turn include chondroitin sulfate chains) may be preferred because ofthz high availability of HA. This may be beneficial in the case of cartilage replacement a repair using the present invention, because the body's normal metabolic pathway for generating cartilage may be able to build directly onto an implanted tyraminc cross-linked T-HA network as will be described
The dityramine cross-linked T-HA network described above has particular utility for producing artificial or synthetic cartilage. Cartilage implants are frequently used in reconstructive procedi res of the head and neck to repair lartilagmous or bony defects secondary to trauma o:: congenital abnormalities. Applications specific to the ear include otoplasty and auricular reconstruction, which are often undertaken to repair cartilaginous defects due to trauma, neoplasm (Le., squamous cell carcinoma, basal cell carcinoma, and melanoma), and co:og
WO 2004/063388 PCT/US20O4/000478
subglottic or txacheal stei .osis. Hie etiology may be traumatic (i.e.? intubation trauma, or tracheotomy) or idioputh.c
Other possibilitie i include chin and cheek augmentation, aad use in ectropion repair of the lower eyelid, in ac dition to numerous craniotacial applications. It should be noted tbat these applications may o ot need cartilage with the exacting mechanical properties of articular cartilage. Inclusion of a cell population of bioactive agents may also be desirable.
One particular ajipHcarion where a croBs-linked network according to tie invention will have substantial utl ity is in the production of an artificial kidney. The kidney filters blood by two mechanist as: one is by size exclusion and the second is by charge exclusion. MEMS devices have be ;n designed for use in artificial kidney devices, which contain precisely defined miao pores that can effectively mimic only the size exclusion characteristics of the kii iney. In a healthy kidney, the charge exclusion related fixation is the result of heparan sutfai proteoglycans present in a basement membrane, which separates two distinct ceU types imp© riant for other kidney related functions. To mimic this charge barrier in the MEMS enguuxan sd artificial kidney, hydrogels can be prepared composed of either heparan sulfaie or hepa tin that are cross-linked via dihydioxypbenyl (dityramioe) links as described herein and pi ovided within the pores of the MEMS device. This heparin'heparan sulfete hydrogel can th in be sandwiched between two hyuluronan derived hydrogels (e.g. T-HA described above) as described herein, and containing one of each of the cell types normally found in a no finally functioning kidney. The central heparin/hepaian sulfate hydrogel provides the • iharge exclusion properties for the device. The outer two hyaluronan hydrogel layers provid s protection from, the immune system and fouling by normal cellular and molecular debris. Inclusion of the two cell types on opposite sides of the filtration barrier provides a cellular component TO its normal physiologic orientation.
In another protoising application, the hydrogels according to the invention can be applied hi developing an artificial pancreas. A problem in development of an artificial pancreas is the short half life of MEMS engineered glucose sensors due to fouling of the detector electrode in vivo. Coating of the surface of these detectors with a hyaloronan hydrogel (e.g. T-HA) as described herein would permit difiusion of the small molecular weight glucose mo lee lies that they are designed to detect while providing protection from Ac immune system and fuling by normal cellular and molecular debris.
In summary, ii will be evident from the foregoing that macromolecules useful as scaffold materials Ibr formation of hydrogels include but are not limited to polycarboxylates

WO 2004/063388 PCT/US2004/000478
(containing free carboxy ate groups), polyamines (containing free primary amine groups), polyphenols (containiag free hydroxyphenyl groups) and their copolymers, examples of which have been desalt ed above. When poryphenols are used, the first step in preparing the network according to tin: invention described above can be omitted because polyphenols already contain multiple or periodic hydroxypheoyl groupa. Otherwise, both porycarboxylates and pc lyamines must have hydroxyphenyl groups added or substituted along their length, prefe rably via the above-described cartiodiimide reaction pathway. The second step in prepaiinj: the network is to cany out an enzyme driven dimerization reaction between two hydro x>pl oxyl groups attached to adjacent iiacroxnolecules (whether pqlycarboxylates, polys mines or polyphenols) in order to provide a cross-linked structure. This step k carried cut using a peroxide reagent (preferably hydrogen peroxide) in the presence of a suitable enzyme (preferably HELP) under metabolic conditions of temperature aridpH,
In the case of U e prefered dityramine cross-linked T-HA network, in the first step the carboxyl groups on hig h. molecular weight hyaluronan (HA) are substituted with tyramine which introduces react ve hydroxypheoyl groups into the HA molecule. This tyramine substitution reaction pi eferably is mediated by the carbocliimidc, 1 -ethyl-3-(3-dimethylaminopropyl)i ^rbodiimide (EDC) with the degroe of tyramine substitution on HA controlled by the mote: ratios and absolute concentrations of tyramine, EDC and HA used in the reaction mix. Ex.« as reagents such as unused tyramiie and EDC are subsequently removed by dialysis, allowing isolation and recovery of high molecular weight tyramiac-substituted HA (T-HA). The percent tyramine substitution within each T-HA preparation is easily calculated by m jasuring: 1) the concentration of tjffamine present in the preparation, which is quanrhated s] •ectrophotometrically based on the unique UV-absorbance properties of tyramine at 275 nm (s( x Example 2 below); and 2) the concentration of total carboxyl groups is the HA preparation, which is quantitated spectrophotometrically by a standard hexuronic acid assay. By this tenqi From. this formulation of T-HA (i.©. 4-6% tyramine substitution) a wide range of biomaterials with a wide range of pbysi
WO 2004/063388 PCT/TJS2004/OOO478
In the cross-liakir g reaction, solutions of T-HA are 1 rings of tyramine resulting in the formation of the dityramine crosslinks. The diryramiae linked structures are fluorescent blue (see Example 2), aprepcrty which is used to both in lage the hydrogels and to quantify the degree of cross-linking within the hydrogels. Since ths cross-linking reaction is enzyme driven, the hydrogels can be formed under physiologic conditions, and therefore can be formed in the presence of included cells or bioactive agent: i, or directly adjacent to living tissue while maintaining cell and tissue viability.
The resulting hydrogels are optically clear with a wide range of physical properties depending on the initia [ T-HA concentration. For example, hydrogels formed from T-HA solutions of 6.25,12.5,25,50 and 100 mg/ml T-HA have been shown experimentally to have physical properties (rigidity, rheology and texture) of a jelly, a gelatin, a dough, a resilient rubber-like compositio 11 (similar to a rubber ball), and a cartilage-like material respectively -see Example 3. These materials have potential applications in a wide range of clinical settings including tissue engineering of both orthopedic (Le. cartilage, bone, tendon, meniscus, intervertebxiU disk, etc) and non-orthopaedic (kidney, liver, pancreas, etc.) tissues, gene and drug deliver;', coating of non-biological device} for in vivo implantation (i.e. glucose sensors, artificial hearts, etc.), wound repair, biosensor design, and vocal chord reconstruction.
Advantageous properties of the hydrogels descrilied herein include the ability to: 1) provide easy charactaization and quality control; 2) integrate with existing tissue matrices; 3) directly incorporate into newly formed matrices,- 4) directly include cells and bioactive factors; 5) maintain biocompatibUity; 6) control bioresoiption; 7) cast easily into complicated anatomical shapes (se: Example 6 below); and 8) exhibit the mechanical properties of native tissues such as articultr cartilage.
Further aspect 3 of the invention will be understood in conjunction with one or more of

WO 2004/063388 PCT/US2004/000478
the following examples, a'hkh are provided by way of illustration.
EXAMPLES Example 1:
Experimental quintities of tyramine-substituted hyjUnronaa hydrogels having dityramine cross-links according to the invention have been prepared as follows. HA is dissolved at 1 mg/ml bajed on hexuronic acid in 250 mM :2-CN-ojorpholino)ethanc3ulfonic acid (MES), 150 mM NsO, 75 mMNaOH, pH 6,5 containing a 10-fold molar excess of tyramine relative to the molar concentration of HA carboxyl groups, Tyramine substitution onto the carboxyl groups is then initiated by the addition of a 10-fold molar excess of EDC relative to the molar concentration of the HA carboxyl groups. A 1/lOth molar ratio of N-hydroxysuccinimide (MIS) relative to the molar amount of EDC is added to the reactions to assist the EDC catalyzed amidation reaction by formation of active esters. Reactions are carried out at room teaperature for 24 hours, after which the maqromolecular fraction is recovered from unread ed small molecular weight reactants such as tyramine, EDC, NHS, and MES by exhaustiv t dialysis versus 150 mM NaCl and then ultrapure water followed by lyophilizatioa After lyophilization, the tyramine-substitijted HA (T-HA) product is dissolved to working concentrations of betweea 5 and 103 mg/ml in PBS (which is a buffer compatible with cell suspension, in vivo tissue contact, and the cross-linking reaction) to provide various concentration preparations depending on the desired rigidity of the final hydrogeL Alternatively, the solvent can be any other suitable solvent besides PBS that will not substantially negatively impact the enzyme activity and that will not interfere with cross-linking reaction via sc active uptake of free radicals generated by the enzyme. Suitable alternative solvents include water, conventional biological tissue culture media, and cell freezing solution (gen wily composed of about 90% blood serum and about 10% dimethyl sulfoxide) Prior to suspension of cells (see Example 5) or contact with tissues in vivo, the T-HA should be filtered through a 0.2 urn filter. Next, tyrjunine-tyramine linking is carried out by adding 10 U/ml of type II horseradish peroxidase (HKP) to each T-HA preparation. Cross-linking is initialed by the addition of a small volume (1-5 ul) of a dilute hydrogen peroxide solution (O.C 12%-0.000l2% final concentration) to yield the final bydrogcl with desired rigidity. For ] (reparation of larger quantities or volumes of a desired hydrogel, quantities of reagents provided in this paragraph could be scaled up appropriately by a person of ordinary skill in the art

WO 2004/063388 PCT/US2004/0004 78
Example 2:
An experiment v as conducted to determine the degree of tyramine substitution (and consequent dityramke cross-linking) for a T-HA macromolecular network according to the invention. Initially, three formulations of (uncrosslinked) tyranxine-substituted hyahironan (T-HA) were prepared «s described above, designated OX, IX or 10X. The OX formulation was prepared using no 1SDC (i.e. containing no carbodiimide), meaning there was no carbodiimide present to mediate the reaction for creating cm amide bond between the NHj group on tyramine and t CO2H group on the HA molecules. Thus, the OX formulation can be considered a control. I be 1X formulation contained a 1:1 stoichiometric ratio of EDC based on the quantity of CO2II groups present on the HA molecules in the reaction mixture. The 10X formulation contained a 10:1 stoichiometric ratio (or 10-fold excess) of EDC based on the quantity of CO2H g coups present on the HA molecules in the reaction mixture. In all three formulations, a stoichiometric excess of tyramine was provided relative to the quantity of CO2H groups on HA. In all three formulations (OX, IX and 10X) the reactants and the appropriate amount of EDC for the formulation were combined in a vial and agitated to facilitate the tyrgmuie- substitution reaction. All three formulations were allowed to react for 24 hours at room temperature, after which the vial contents were dialyzed to remove unreacted tyramine me lecules, EDC and acyiuiea (EDU) byproducts of the reaction. These molecules ware easily separated from HA and any formed T-HA molecules through dialysis due to the relatively small size of tyramine, EDC and EDU compared to macromolecular HA. Once unreacted tyramine and EDC were removed, the remaining contents for each formulation were anal; /zed to determine the rate of tyranoiine substitution relative to the total number of available C02H sites present on HA molecules.
Tyramine exhi >its a UV absorbancc peak at 275 jam, making the degree of tyramine substitution easily deti >ctible against a tyramine calibration curve. Based on UV-spectroscopic analysis of the above three T-HA formulations, it was discovered that the HA-tyramine substitution :."eaction carried out with no EDC present (formulation OX) resulted in substantially zero tyra mine substitution onto the HA molecules. This confirmed the importance of using a carbodiimide reaction pathway in the tyramine substitution reaction. However, the tyramin 5 absorption in the T-HA formulation prepared using a 1:1 EDC:CO2H stoichiometric ratio ii the tyramine substitution reaction (formulation IX) resulted in a tyramine substitution rate of about 1.7% relative to all a^/ailable CO2H groups on the HA

WO 2004/063388 PCT/U52f>«W000478
chains. The 10X formulilion (10:1 EDCiCOzH ratio) resulted in about a 4.7% substitution raie.
Subsequently, hy irogen peroxide and horseradish peroxidase (HRF) were added to each of the three dialyze i HA/T-HA formulations (OX, IX and 10X) at 5 mg/mL and the resulting formulations w ere allowed to react to completion. After reaction in th« presence of peroxide and HRP, it w The dityramiue structure exhibits a blue fluorescence on exposure to UV light. The products of each of the above formulations were exposed to UV light to detect the presence of dityramine cross-bn is. As expected, both the IX and 10X hydrogels exhibited blue fluorescence (the 10X hydrogel fluorescence being more intense than that of the IX hydrogel), while the; 01C formulation exhibited no blue fluorescence at all. This confirmed the presence of dityramuK cross-links in both hydrogels, and', that the occorxence of dityramiae in the more rigid hydrogd (10X) was greater than in the less rigid hydrogel (IX).
The overall result was that the importance of the i^arbodiimide-mediated reaction pathway was demonst "ated, and it was confirmed that the relative rigidity of a hydrogel formed from a crosu-liciked T-HA network according to ijhe invention is proportional to the degree of diryraome cross-Unking, which is in turn proportional to the degree of tyramine-substiturion onto HA. It was quite a surprising and unexpected result that even a 1.7% tyranune-substitution rate (and subsequent cross-linking rate to form dityremino links) provided a suitably fii m T-HA gel (or hydrogel) according to the present invention. A 4.7% substitution (and wo* i-lmking) rate resulted hi even a fiimer T-HA geL Also surprising was that a ten-fold stoichic metric excess of carbodiimide (EDC) relative to the quantity of carboxylic acid group J present in the reaction mixture (formulation 10X) resulted in only about a 4.5-4,7% tyra: nine substitution rate, yet stable and cohesive tyramine cross-linked T-

WO 2004/063388 PCT/US2004/000478
HA networks were no net heless achieved
This means fort t ie majority of the carboxylic acid groups on the HA. molecules are uosubstiiuted and not tyt amine cross-linked, essentially remaining the same as in the native HA molecule, yet the r« .ulting network is a cohesive and stable hydtogel Therefore, when used as a cartilage subst: tute in vivo, because a majority of the HA molecules in the invented T-HA network or gel an: essentially unaltered compared to HA in normal cartilage, it is believed titat the body's native metabolic pathways (aided or unaided by cells provided within the T-HA aetwoik) may recognize the invented network as native biologic material, and will be able to carrj out ordinary synthesis and metabolism functions with, respect thereto. In addition, it h noted that HA is a highly ubiquitous material in the body, and is uon-immunogenic in hi mans. As a result, it is believed trie invented cross-linked macromokcular netwoi k, comprised a majority of unaltered native HA, will have substantial application in a wide vs riety of tissue engineering applications where it is desirable or necessary to provide sy athetic tissue in a human body. Tliis represents a significant advance over the state of the an Therefore, quite surprisingly, a high degree of tyramine substitution, e.g. greater than about 10-20%, may be undesirable; the above described experiments demonstrated that such high degrees of substitution are unnecessary to provide a suitable T-HA network. Preferab y, a dihydroxyphenyi (e.g. dityrafliine) cross-linked potycarboxylate (e.g. HA) network according to the invention has ahydroxyphenyl (tyramine) substitution rate of less than 50, preferably less than 40, preferably less than 30, preferably less than 20, preferably less than 15, preferably less than 10, preferably less than 9, preferably less than 8, preferably less than 7, preferably less than 6, preferably less than 5, percent based on the total quantity of CO2H groips present on thepolycarboxylate (HA) molecules.
Example 3:
Conventional!}', it has been believed that natural cartilage exhibits its viscoelastk: properties and its ability to resist deformation and absorb compressive loads principally as a result of the repulsive forces between negatively charged SO42* groups on adjacent chondrokm sulfate chi tins present in the aggrecan matrix. An experiment was performed to determine the efficacy of a macromolecular network according to the invention consisting only of dityramine crc ss-linked hyaluronan molecules (i.e. no aggrecan or chondroitin sulfate) to resist deformation and absorb compression compared to natural cartilage despite the absence of SO4* g roups. A formulation of uncross-Jinked T-HA was prepared and

WO 2004/063388 PCT/US2004/(m0478
purified as in Example 1 having a tyramine substitution rate of about 5%. From this T-HA formulation, five difieral T-HA concentrations wereprepstred;
Concentration 1: 6.25 mg T-HA / mL PBS Concentration 2: 12.5 mg, T-HA / mLPBS Concentration 3; 25 mg T-HA / mL PBS Concentratioc. 4: 50 mg T-HA / mL PBS Concentratiori 5: 100 mg T-HA / mL PBS
Each of the aboi e preparations was then reacted in the presence of hydrogen peroxide and horseradish peroxic ase, also as in Example 1, to form dityramine cross-links between the T-HA molecules aid pi ovide respectively Hydrogels 1,2,3,4 and 5. Each of these five hydrogels was found, si irprisingly and unexpectedly, to be; a stable and substantially coherent material with the physi al properties of each hydrogel varying relative to the concentration of T-HA in the preparatio i from which it was made. For example, qualitatively Concentration 1 resulted in Hydrogei. 1 oaving rigidity and rheological properties comparable to that of Vaseline or jelly, the hydrogel was stable and coherent yet could be caused to flow or spread on, application of en ex rental force, e.g. from a spatula or other conventional tooL Hydrogel 1 exhibited excellent adl esive properties making it an ideal candidate for a nonallergenic coating material for sugical instruments during surgery, e.g. ophthalmologic surgery. Hydrogel 2 was mare 1 igid than Hydrogel 1 due to the greater concentration of T-HA in the preparation from wliic'i it was made, and the consequent predicted decrease in intramolecular cross-linking and is.cn ase in. intermolecular cross-Unkinj; associated with increased T-HA concentration. Hydro j ;el 2 exhibited rheological and rigidity properties characteristic of gelatins, with a degree of viscodastic reboundability on external loading. On greater loading, Hydrogel 2 was found to break up into smaller pieces imitead of flowing, also characteristic of a gelatinous matsrii iL Hydrogel i had the properties find consistency of a dough or malleable paste, also not flowing on application of an external loading force. This material also exhibited subs tan dally greater viscoelastic propertks compared to Hydrogels 1 and 2. Hydrogel 4 was a tig] ily rigid and coherent gel that strongly resisted breaking up on application of an cscte nal loading force. Hydrogel 4 was a highly resilient rubber-hlce composition that actu flly generated substantial springing force upon sudden compression (e.g. dropping ontc th e floor). This ability of Hydrogel 4 to generate such a springing force

WO 2004/A63388 PCT/US2004/00047S
in response to a sudden The compressive mechanical properties of the five hydrogels were determined as described in the prixe ling paragraph. Load data was normalized by sample cross-sectional area (39.6 mm7) to co tnpute stress. The equilibrium stress was plotted against the applied strain for each mat art; il formulation. The aggregate modulus at each step was defined as the equilibrium stress div ided by the applied strain. For each material, the aggregate modulus was defined as the sic pe of the equilibrium stress-strain data in the most linear range. The results for the confined compression tests are shown in Fig. 4. All five hydrogels were testable in confined compression, and demonstrated characteristic stress relaxation responses typical of biphasic mi iterials (such as cartilage). The aggregate moduli for the 625 mg/ml and 12.5 mg/ml T-HA hydrogels were 1-2 wders of magnitude tower than articular cartilage.

WO 2004/063388 PCT/US20O4/OOO478
The 25 mg/ml T-HA hydrogel displayed an aggregate modulus of approximately half of the reported values for ertirolar cartilage. The 50 and 100 mjj/ml T-HA hydrogels displayed aggregate moduli, whi cartilage.
Based on the a'xwe experiments it was surprisingly and unexpectedly discovered that a dityramiae cross-linl ;ed hyaluronan network will produce a coherent hydrogel material whose rigidity- and otb sr physical (rheological) properties} can be tuned by varying the T-HA concentration prior to cross-linking the tyramine groups to suit a particular application. The coherence and elastic jroperties of these hydrogels was observed even absent any (or substantially any) SO*2 groups in the network to supply the charge-to-charge repulsive forces to generate the materi il's compression resistance and elasticity. This was a highly surprising and unexpected result that may have substantial positive consequences in tissue engineering applications. Hyaiwcnan is a highly ubiquitous and non-immunogenic molecule found in humans. Therefore, lydrogels consisting of dityramine cross-linked hyaluronan networks may provide very suit able tissue replacement materials that can be implanted within a human body, whose rigidity A number of methods of preparing hydrogels such as those described in Example 3 have been developed to cast or form the hydrogel into a predetermined three-dimensional shape. This is import ant for myriad tissue engineering implications where it is necessary to provide artificial tissi te material to fill a native tissue defect or void within a patient
A first methoi 1 is to employ an in situ forming technique where the hydrogel is formed in place, i.e. in p«:iti )n and in the shape of its final application and structure. The in situ formation method ha i been carried out experimentally as follows. .Tyramine-substituted

WO 2004/063388 PC17US2004/000478
hyaiuronan (T-HA) was prepared via the caftoo'nmite-mediated pathway described herein. Following dialyzafioc. to remove unreactedtyramiae, EDC, NHS, etc, and dissolution at the desired concentration in PBS (see Example 1 above), a snuill quantity of horseradish peroxidasc enzyme was added to the T-HA Kquid preparation to form a first solution. This first solution was provided into a laboratory container (to simulate an in vivo situs) having a specific interior geomet y. Subsequently, a second solution was prepared containing very dilute hydrogenperoidde (0.012%-0.00012% final concentration), A small volume of this second solution relative to the first solution was then injected into the container already containing the first sola tion to initiate the dilyramine crosj-linking reaction to yield the hydrogei. HydrogeLs p epared by this technique have been prepared having varying rigidity and rheological properties as described above in Example 3, and conformed well to the interior surface contoui of the container in which they were formed.1 Because the principal reagents (H2O2, hyaluronaa and peroxidase) are either nouallergenic or diffusible molecules, and because the cross-linking reaction proceeds under metabolic conditions of temperature and pH, this technique can be performed in vivo ait a surgical situs hi a patient as a surgical procedure to produce a defect-conforming hydrogei This method is particularly attractive tor reconstructive fac'u 1 surgery in which the uncross-linked T-HA preparation (with peroxidase) can be injc cted and manipulated subcutaneoiisly by the surgeon to produce the desired racial contours and then the hydrogei subsequently cross-linked by injection of a small volume of the: hj drogen peroxide solution.
A second raeth 3d is a porous mold technique and is suitable for forming hydrogels into more complex thr ^-dimensional structures. In this technique a porous hollow mold is first cast conforming t > the shape and contour of the intended final structure. For illustration, a mold can be prepare 1 having an interior surface in a cuboid shape if a cuboid shaped bydnjgel were desired The mold can be prepared or cast via conventional techniques from conventional porous n taterials, e.g. plaster of pans, porous or sintered plastics or metals, etc. Io a particularly prcfta red embodiment the mold is prepared using a cellulosic dialysis membrane. The first
WO 2004/063388 PCT/CS2004/00047S
tinting occur from the oirtside inward to produce the finishsd hydrogel shape, and a certain degree of trial and error: nay be required to determine optimal or sufficient immersion times in the peroxide bath. Daetmination of these time periods is within the skill of e. person, having ordinary skill in i he art. Successfully completed three-dimensional hydrogel shapes have been prepared in Is boratory bench experiments via this poions mold technique.
A third method is a freeze-thaw technique that is suitable for casting hydrogels according to the inventi >a in highly intricate predetermined three-dimensional shapes, e.g. having internal folds su;h as a human ear. In this technique, a mold is prepared from asoft or malleable material sv ch as a polymeric material having a low glass transition temperature, e.g. below -80°C. The preferred mold materials are sflicones having low glass transition temperatures, such as pDlydimethylsiloxane whose glass transition temperature is about -127"C, however other suitably low glass transition (e.g. below -80DC) sflicones, as weU as other polymers, can be used. The silicone (preferred matsrial) is first prepared such that h has an inner mold cuvi y conforming to the surface shape, contour and volume of a desired hydrogel part via any c onventional or suitable technique (Le. press-molding, carving, etc). First and second solutions are prepared as above, and the first solution is provided into the inner mold cavity of tr e silicone mold. The now-filled silicone mold is then cooled to about -80°C by contacting v ith solid COj (dry ice). Because the first solution is principally water, it freezes into a solid i:e form conforming to the shape and contour of the inner mold surface. However, the silicone mold, having a glass transition temperature below -8Q°C, remains soft and malleable and the solid ice form of the first solution is easily removed. Because the first solution expands as it freezes, suitable mechanical hardware should be used to ensure the silicone mold does no: deform or expand as the solution freezes. Preferably, port holes are provided in the mold 10 allow for expansion and discharge of the first solution as it expands during the freezing pr jcess.
Once the solid ice form of the first solution has teen demolded, minute defects or flaws in the three-din ensional structure can be repaired by carving with a suitable tool, and more of the liquid firs t solution can be added to fill surface voids, which liquid instantly freezes on contact wi)h the solid ice form. Also, the ice form can be placed back on the dry ice surface if desired » ensure uniform temperature and freezing of any added first solution material. Once this tt ree-dimensional shape of the ice form has been perfected, it is immersed in a liquid peroxide solution to initiate thawing of the frozen water and diryraminc cross-lmkiiig from th; outside-in. This is possible do to the rapid kinetics of the cross-linking

WO 2004/063388 PCT/US2004KKKI478
reaction. Cross-linking is. determined to be complete once the last remaining frozen water has melted at the center of ti e forming hydrogel form, which can be easily observed because the forming hydrogd is subiitantiaUy clear.
Very successful experiments have been performed according to this freeze-thaw technique to produce a s olid bydrogel according to the invention in the shape of a human ear. Other structures that coi dd be formed by this method, such as intervertebral discs, meniscus, etc. will be evident to tl ose skilled in the art. It should be noted in ibis freeze-thaw technique, the threshold glass transition temperature of -80°C for the mold material is selected to correspond i oughly with the surface temperature of solid CO2 (dry ice), to ensure the mold material do es not become brittle when the first solution is frozen to produce the solid ice form. HOWCVT, if another cooling material, other than CO2 is used, then the threshold glass transttk m temperature for suitable mold materials may be adjusted . accordingly.
For the three: m 3hods of hydrogel formation described above, the first solution contained both the pen ixidase and T-HA, while the second solution contained the peroxide. While ii may be possit ie to switch the peroxidase and peroxide in the first and second solutions respectively, it is less preferred to provide the peroxide in the first solution with the T-HA. This is becaus*: once the peroxide, peroxidase and T-HA are combined, the T-HA rapidly begins to form a cross-linked macromolecular network. If the peroxidase (which is a macromoleailar mole* :ule) is not already uniformly distributed with the T-HA it may be unable or substantially hindered from diffusing through the pore structure of the forming hydrogel to facilitate 1 nifbrm cross-linking throughout tlie entire T-HA/peroxide solution. The result could be no n-unifbrm and/or incomplete crossi-linkmg of the T-HA and a non-uniform hydrogeL Cc aversely, the relatively small peroxide molecule (hydrogen peroxide is only one oxygen atom larger than water) can diffuse through the hydrogel pore structure with relative ease, resulting, in a uniform hydrogel structure,
In addition, th; macromolecular size of the peroxidase allows it to be similarly retained as the T-EiA within porous molds that are only porous to small molecular weight peroxides which easil y and uniformly diffuse through both the molds and newly forming macromolecular netw arks (Le. hydrogels). For these reiisons it is preferred to start with the peroxidase uniformly distributed with the T-HA in the first solution, and to provide the peroxide separately in the second solution.
A fourth metfc od is an alternating sprayed or brushed layering technique. The first

WO 2004/063388 PCT/US2004/0IMU78
solution is prepared as' d All four of toe jbove techniques have been described with respect to dityramine cross-linked hyalurone n, however it will be understood tliat other combinations within the scope of the preseni: invention (other dmydroxyphenyl cross-linked macromolecules, such as porycarboxylates, porj amines, polyhydroxyphenyl mobiles and copolymers thereof) can be molded via the above :echniques.
iF-yatwpl1'- V
Rat chondrocytes were embedded hi (cross-linked) T-HA hydrogels to measure then-ability to survive the dispersed with the T-HA and peroxidase, followed by introduction of the peroxide-containing second solution to initiate dityrmnine cross-Unking. The chondrocyte-embeddod 1.7% and 4.7% T-HA hydrogeb; exhibited uniformfy distributed chondrocytes with th<: optical clarity of the gels allowing visualization throughout gel glucose utilization w js used as an indicator ccu viability after cross-unking to form hydrogels chondro cytes are voracious with respect tc consumption depleting>
WO 2004/063388 PCT/US20rt4/000478
medium of glucose in le is than 24 hows. The results showed that chondrocytes embedded in T-HA hydrogels showed. essentially the same glucose consumption F^6 over 24 D0WS w the same chondrocytfs Fluorescent isaajes of frozen sections of T-HA hydrogels containing both chondrocytes and cartil ige tissue were also generated, HA samples from both the hydtogel scaffold and cartilage n latrix were visualized by fluorescent staining with biotinylated HA binding protein (b-HAI5P) reagent while cell nuclei were visualized with standard DAPI stain. The b-HABP r« gent is prepared from purified canilage aggrecan (the Gl domain only) and link protein, and recognizes and irreversibly binds to stretches of native HA equivalent to those nor anally bound by aggrecan and Hnk protein in cartilage. The results showed a more intense staining of the T-HA hydtogel wi'h b-HABP than the cartilage as the hyaluronan in the tiijsu z is already occupied by native agjjrecan and link protein. No visible distinction could be se ;n between the T-HA scaffold of tie hydrogel and the matrix of suspended cartikge tis sue suggesting seamless integration. These results demonstrated the feasibility of maintaining the viability of chondrocytes during the hydrogel cross-linking reactions, and the a'bili ty of the hydrogel to integrate seamlessly into existing cartilage matrix, both of which are advantageous for application to cartikge repair. The results also demonstrated that sufl idem stretches of the T-HA remain chemically unaltered, and available for binding by newly s synthesized aggrecan and link protein in situ. The results also demonstrated that oxygen, carbon dioxide, glucose and insulin are diffusable through the T-HA hydrogels according to the invention at a rate that is not limiting to chondrocyte metabolism, which is important not only to the development of cartilage substitutes but to other applications sue i as glucose sensor design and development of an artificial kidney. In order to inc tude cells such as chondrocytes hi hydrogels molded into intricate anatomical shapes usi ag the freeze/thaw technique described hi Example 4, it is desirable that the enzyme driven cr
WO 2004/1*63388 PCT/liS2lHMWWU7J,
Although the above-described embodiments consti rate the preferred embodiments^ it ■will be understood tliaf raiioua changes or modifications can be made thereto vnthout departing from the spin: and the scope of the present invention as set forth in the appended

1. A macromolecular network comprising
wherein R\ and R2 each comprises a structure selected from the group consisting of polycarboxylates, polyamines, polyhydroxyphenyl molecules, and copolymers thereof, and wherein Ri and R2 can be the same or different structures.
2. A macromolecular network according to claim 1, wherein Ri is a polycarboxylate.
3. A macromolecular network according to claim 1, wherein Ri comprises a structure
selected from the group consisting of glycosaminoglycans.
4. A macromolecular network according to claim 1, wherein Ri comprises hyaluronan.
5. A macromolecular network according to claim 1, wherein Ri comprises a structure
selected from the group consisting of chondroitin sulfate and dermatan sulfate.
6. A macromolecular network according to claim 1, wherein Ri comprises aggrecan.
7. A macromolecular network according to claim 1, said network comprising
polycarboxylate molecules that have been substituted with a hydroxyphenyl compound, wherein
at least one dihydroxyphenyl linkage is formed between two hydroxyphenyl groups attached
respectively to adjacent polycarboxylate molecules.
8. A macromolecular network according to claim 7, said hydroxyphenyl compound being
tyramine or tyrosine.
9. A macromolecular network according to claim 7, said polycarboxylate molecules
having a hydroxyphenyl compound substitution rate less than 50 percent based on the molar

quantity of CO2H sites present on said polycarboxylate molecules.
10. A macromolecular network according to claim 1, comprising a plurality of tyramine-
substituted hyaluronan molecules, at least two adjacent hyaluronan molecules being linked by a
dityramine linkage.
11. A macromolecular network according to claim 10, having a tyramine substitution rate
on said hyaluronan molecules of less than 10% based on the molar quantity of CO;>H sites
present on said hyaluronan molecules.
12. A macromolecular network according to any of claims 1-11, further comprising a
population of viable living cells and/or bioactive factors within the macromolecular network.
13. A hydrogel comprising a macromolecular network according to any of claims 1-12.
14. A hydrogel according to claim 13, having rigidity and rheological properties that are
tunable to be those of a jelly, a gelatin, a dough, a resilient rubber-like composition or cartilage.
15. A hydrogel according to any of claims 13-14, provided as synthetic or artificial
16. A method of making a macromolecular network comprising the steps of providing a
macromolecular species selected from the group consisting of hydroxyphenyl-substituted
polycarboxylates, hydroxyphenyl-substituted polyamines, other polyhydroxyphenyl molecules,
and copolymers thereof, and forming at least one dihydroxyphenyl linkage between two
hydroxyphenyl groups attached respectively to adjacent ones of said macromolecular species.
17. A method according to claim 16, said macromolecular species being a
hydroxyphenyl-substituted polycarboxylate that is prepared by carrying out a chemical reaction
between a) carboxylic acid functional groups on polycarboxylate molecules selected from the
group consisting of hyaluronan molecules and chondroitin sulfate molecules, and b) primary
amine functional groups on a second species selected from the group consisting of tyramine and

tyrosine, to thereby provide tyramine- or tyrosine-substituted hyaluronan or chondroitin sulfate molecules, wherein the tyramine or tyrosine substituents on these polycarboxylate molecules impart hydroxyphenyl groups thereto.
18. A method according to claim 17, said second species being tyramine and said
polycarboxylate molecules being hyaluronan molecules, the resulting macromolecular network
being a dityramine cross-linked network of tyramine-substituted hyaluronan molecules.
19. A method according to claim 17, said chemical reaction being carried out via the
following pathway.

a) reacting a carboxylic acid group on said polycarboxylate molecules with a
carbodiimide to form an O-acylisourea-substituted polycarboxylate, and
b) reacting said O-acylisourea-substituted polycarboxylate with the primary amine group
on said second species to form said hydroxyphenyl-substituted polycarboxylate.

20. A method according to claim 19, said carbodiimide being l-ethyl-3-(3-
21. A method according to claim 19, step (a) being carried out in the presence of an
active ester forming catalyst.
22. A method according to any of claims 16-21, further comprising forming said
dihydroxyphenyl linkage by reacting said hydroxyphenyl groups with a peroxide in the presence
of an enzyme to yield adjacent hydroxyphenyl free radical species, said free radical species
dimerizing via a dimerization reaction to yield said dihydroxyphenyl linkage.
23. A method of making a hydrogel comprising the steps of:

a) providing a first solution comprising either a peroxidase enzyme or a peroxide but not
both, and also a macromolecular species selected from the group consisting of hydroxyphenyl-
substituted polycarboxylates, hydroxyphenyl-substituted polyamines, other polyhydroxyphenyl
molecules, and copolymers thereof;
b) providing a second solution comprising the one of said peroxidase enzyme or peroxide

not provided in said first solution; and
c) combining said first and second solutions to initiate dihydroxyphenyl cross-linking to
form said hydrogel.
24. A method according to claim 23, further comprising providing a non-porous mold
having an inner mold cavity conforming to the shape, contour and volume of a desired hydrogel
part, and combining said first and second solutions within said inner mold cavity to form said
hydrogel in the shape thereof.
25. A method according to claim 23, further comprising the steps of:

d) providing a mold made from porous material having an inner mold cavity conforming
to the shape, contour and volume of a desired hydrogel part;
e) providing said first solution into said inner mold cavity of said mold; and
f) immersing said porous mold having said first solution therein in a bath of said second
solution to thereby initiate said dihydroxyphenyl cross-linking and formation of said hydrogel.
26. A method according to claim 23 further comprising the steps of:
d) providing a mold made from a pliable material having an inner mold cavity
conforming to the shape, contour and volume of a desired hydrogel part;
e) providing said first solution into said inner mold cavity of said mold;
f) cooling said mold to freeze said first solution within the inner mold cavity thereof to
provide a solid ice form in the shape of said inner mold cavity; and
g) immersing said solid ice form in a bath of said second solution to thereby initiate
thawing of frozen water and dihydroxyphenyl cross-linking to form said hydrogel.
27. A method according to claim 23, said second solution further comprising said
macromolecular species, the method further comprising alternately applying layers of the first
and second solutions to a situs to thereby initiate said dihydroxyphenyl cross-linking and
formation of said hydrogel.

A dihydrophenyl XXX cross-linked macromolecular network is provided that bis in articles XXX and XXX and XXX application such as a XXX synthesis catridge.The ntework is made by first providing a polyamines or polycarbomate myclonucleotide xxx a secquirity of amine or carbonyle and respectively structured along the length of the molecule.re acting thus macroXXX with a XXX compound having a free carboxylic acid group in the case of a polycrane or a XXX primary smine group molecule of a polycarbonate and subsdrung the hydroxyphenyl compound onto the macromelecule via ii embodiment obtained XXX pathway to provide a hydroxyphenyl substituned macromolecules.This macromolecule is then linked to other such macro XXX an enzyme catalysed dimenzatoic reaction between two hydroxy groups attached respectively in different mesurement under XXX condition of tempareture and pH.In a prefered embodiment the thereto molecules network is made up hydramine substutents XXX molecule that are visited by dimethyl loads to provide a stable cohherent hydrogen with detail physical propetides.A method of preparing such a network is also provided.


Patent Number 214247
Indian Patent Application Number 01504/KOLNP/2005
PG Journal Number 06/2008
Publication Date 08-Feb-2008
Grant Date 07-Feb-2008
Date of Filing 01-Aug-2005
Applicant Address 9500 EUCLID AVEUNE, CLEVELAND, OH 44195, U.S.A.
# Inventor's Name Inventor's Address
PCT International Classification Number C12Q
PCT International Application Number PCT/US2004/000478
PCT International Filing date 2004-01-09
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/439 201 2001-01-10 U.S.A.