New strategy for chemical modification of hyaluronic acid:
`Preparation of functionalized derivatives and their use in
`the formation of novel biocompatible hydrogels
`
`Paul Bulpitt, Daniel Aeschlimann
`Division of Orthopedic Surgery, University of Wisconsin, H5/301 Clinical Science Center, 600 Highland Avenue,
`Madison, Wisconsin 53792
`
`Received 2 September 1998; revised 4 January 1999; accepted 3 March 1999
`
`Abstract: Biodegradable materials for spatially and tempo-
`rally controlled delivery of bioactive agents such as drugs,
`growth factors, or cytokines are key to facilitating tissue
`repair. We have developed a versatile method for chemical
`crosslinking high-molecular-weight hyaluronic acid under
`physiological conditions yielding biocompatible and biode-
`gradable hydrogels. The method is based on the introduc-
`tion of functional groups onto hyaluronic acid by formation
`of an active ester at the carboxylate of the glucuronic acid
`moiety and subsequent substitution with a side chain con-
`taining a nucleophilic group on one end and a (protected)
`functional group on the other. We have formed hyaluronic
`acid with amino or aldehyde functionality, and subse-
`quently hydrogels with these hyaluronic acid derivatives
`and bifunctional crosslinkers or mixtures of the hyaluronic
`acid derivatives carrying different functionalities using ac-
`tive ester- or aldehyde-mediated reactions. Size analysis of
`the hyaluronic acid derivatives showed that the chemical
`
`modification did not lead to fragmentation of the polysac-
`charide. Hydrogels formed with hyaluronic acid derivatized
`to a varying degree and crosslinked with low- or high-
`molecular-weight crosslinkers were evaluated for biode-
`gradability by digestion with hyaluronidase and for biocom-
`patibility and ectopic bone formation by subcutaneous im-
`plantation in rats. Several hydrogel formulations showed
`excellent cell infiltration and chondro-osseous differentia-
`tion when loaded with bone morphogenetic protein-2 (BMP-
`2). Synergistic action of insulin-like growth factor-1 with
`BMP-2 promoted cartilage formation in this model, while
`addition of transforming growth factor-b and BMP-2 led to
`rapid replacement of the matrix by bone. © 1999 John Wiley
`& Sons, Inc. J Biomed Mater Res, 47, 152–169, 1999.
`
`Key words: hyaluronic acid; crosslinking; tissue repair; cy-
`tokine delivery; bone formation
`
`INTRODUCTION
`
`In orthopedic surgery, defects in articular cartilage
`present a very complicated treatment problem be-
`cause of the very limited capacity of cartilage to repair
`spontaneously. The failure of regenerating persistent
`hyaline cartilage by surgical procedures prompted in-
`vestigators to attempt repair using biological strate-
`gies.1 Repair has been induced by transplantation of
`culture-expanded autologous chondrocytes2 or by re-
`cruitment of mesenchymal stem cells from the syno-
`vium using chemotactic and mitogenic factors.3 The
`shortcoming of both strategies is the difficulty in sta-
`bly anchoring the repair-inducing factors, whether
`cells or growth factors, within the lesion. Hyaluronic
`acid (HA) is a good candidate for the development of
`
`Correspondence to: D. Aeschlimann
`Contract grant sponsor: Orthogene, Inc.
`
`© 1999 John Wiley & Sons, Inc.
`
`CCC 0021-9304/99/020152-18
`
`novel biomaterials for local delivery of cells and bio-
`active factors because of its unique physicochemical
`properties and its excellent biocompatibility and bio-
`degradability.
`Hyaluronic acid, or hyaluronan, is a natural poly-
`saccharide that is most abundant in cartilage and in
`the vitreous.4 HA plays a key structural role in the
`organization of the cartilage extracellular matrix as an
`organizing structure for the assembly of aggrecan, the
`large cartilage proteoglycan.5 The highly negatively
`charged aggrecan/HA assemblies are largely respon-
`sible for the viscoelastic properties of cartilage by im-
`mobilizing water molecules. HA is unique among gly-
`cosaminoglycans with respect to not being covalently
`bound to a polypeptide. HA is also unique in having
`a relatively simple structure of repeating nonsulfated
`disaccharide units composed of D-glucuronic acid and
`N-acetyl-D-glucosamine (Fig. 1). The molecular mass
`of HA in extracellular matrices is typically several mil-
`lion Daltons. Besides its structural function in extra-
`cellular matrix assembly, HA plays pivotal roles in
`
`ALL 2093
`PROLLENIUM V. ALLERGAN
`IPR2019-01505 et al.
`
`

`

`IN SITU POLYMERIZABLE HYALURONIC ACID HYDROGEL MATERIALS
`
`153
`
`Figure 1. Structure of HA.
`
`promoting cell motility and differentiation in devel-
`opment and wound healing.4,6 Blocking the interac-
`tion of HA with cell-surface receptors, i.e., isoforms of
`CD44, in prechondrogenic micromass cultures from
`embryonic limb bud mesoderm inhibits chondrogen-
`esis, indicating that the establishment and mainte-
`nance of a differentiated chondrocyte phenotype is at
`least in part dependent on HA and HA–receptor in-
`teractions.7
`Based on its unique rheological properties, HA is
`currently being used clinically in viscosupplementa-
`tion and viscosurgery.4,8 Viscous solutions of high-
`molecular-weight HA and its salts are being used in
`therapy for arthropathies by intraarticular injection,9
`to promote wound healing in various tissues, and as a
`surgical aid in eye and middle-ear surgery. More re-
`cently, HA has been used in derivatized and/or
`crosslinked form to manufacture thin films which are
`used to create tissue separations.10 Extensive efforts
`have been made by various laboratories to produce
`derivatives of HA with unique properties for specific
`biomedical applications. Most of the developments
`have been focusing on the production of materials
`such as films or sponges for implantation and the sub-
`stitution of HA with therapeutic agents11–13 for de-
`layed release and/or prolonged effect. In fact, the half-
`life of pharmacological compounds has been shown to
`be drastically increased when delivered systemically
`as a HA conjugate.11,12 Strategies for modification of
`HA have included esterification of HA,14,15 acrylation
`of HA,16 and crosslinking of HA using divinyl sul-
`fone17 or glycidyl ether.18 However, the modified HA
`molecules show altered physical characteristics such
`as decreased solubility in water, and/or the chemical
`reaction strategies used are not designed for crosslink-
`ing of HA under physiological conditions, and thus
`cannot be used to polymerize a biodegradable matrix
`in situ which would be desirable in resurfacing of ar-
`ticular cartilage.
`
`The introduction of functional groups on HA, e.g.,
`amino groups, which could be used for further con-
`venient coupling or crosslinking reactions under mild
`physiological conditions, is a subject of great interest.
`Previous methods have produced a free amino group
`on high-molecular-weight HA by alkaline N-deace-
`tylation of its glucosamine moiety.19,20 However, con-
`comitant degradation of HA via b-elimination in the
`glucuronic acid moiety was observed under the harsh
`reaction conditions needed. An early report claimed
`that carbodiimide-catalyzed reaction of HA with gly-
`cine methyl ester, a monofunctional amine, led to the
`formation of an amide linkage.21 This, however, has
`been proven in a number of studies not to be the
`case.22,23 Under mildly acidic conditions the unstable
`intermediate O-acylisourea is readily formed, which
`in the absence of nucleophiles, rearranges by a cyclic
`electronic displacement to a stable N-acylurea24 (Fig.
`2). This O fi N migration of the O-acylisourea also
`occurs when the nucleophile is a primary amine23 and
`any amide formation that does occur is insignificant,
`as reported by Ogamo et al.22 The carbodiimide-
`mediated introduction of a terminal hydrazido group
`on HA with a variable spacer has recently been
`achieved and has led to the ability to conduct further
`coupling and crosslinking reactions.13,25
`In this study, we have developed methodology for
`introducing side chains into HA by carbodiimide-
`mediated coupling of primary or secondary amines to
`the carboxyl group of the glucuronic acid moiety us-
`ing an active ester intermediate. We demonstrate that
`by “rescuing” the active O-acylisourea by formation of
`a more hydrolysis-resistant and nonrearrangable ac-
`tive ester intermediate, the coupling of primary
`amines to HA is possible. Using this methodology, we
`have generated HA derivatives carrying different
`functional groups for subsequent crosslinking to form
`hydrogels. Characterization of these hydrogels in dif-
`ferent biological assays established that novel biocom-
`
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`

`154
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`BULPITT AND AESCHLIMANN
`
`patible and biodegradable materials are formed with
`these HA derivatives.
`
`MATERIALS AND METHODS
`
`Synthesis of HA derivatives
`
`General procedure of carbodiimide/HOBt reactions
`with HA
`
`Sodium hyaluronate (100 mg, 0.25 mmol; MW > 1 × 106;
`Genzyme Pharmaceuticals, Haverhill, UK) was dissolved in
`
`H2O at a concentration of 3 mg/mL. To this solution was
`added a 30-fold molar excess of an amine or hydrazide (pKa
`3–8.5; 7.5 mmol), i.e., ethylenediamine, adipic dihydrazide.
`The pH of the reaction mixture was adjusted to 6.8 with 0.1
`M NaOH/0.1M HCl. 1-Ethyl-3-[3-(dimethylamino)propyl]-
`carbodiimide (EDC) (192 mg, 1 mmol; Aldrich Chemical
`Co.) and 1-hydroxybenzotriazole (HOBt) (135 mg, 1 mmol;
`Fluka Chemical Corp.) was dissolved in dimethylsulfoxide
`(DMSO)/H2O (1:1, 1 mL). After mixing, the pH of the reac-
`tion was maintained at 6.8 by the addition of 0.1M NaOH
`and the reaction was allowed to proceed overnight. The pH
`was subsequently adjusted to 7.0 with 0.1M NaOH and the
`derivatized HA exhaustively dialyzed (Spectra/Por RC 2,
`
`Figure 2. Strategy for coupling of different amines to HA. Generated aldehyde- and amine-functionalized HA derivatives
`are listed.
`
`

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`IN SITU POLYMERIZABLE HYALURONIC ACID HYDROGEL MATERIALS
`
`155
`
`MW cutoff 12–14,000; Fisher Scientific) against distilled
`H2O. NaCl was added to produce a 5% w/v solution and the
`modified HA was precipitated by addition of 3 vol equiva-
`lents of ethanol. The precipitate was redissolved in H2O at a
`concentration of approximately 5 mg/mL and the purified
`product was freeze-dried and kept at 4°C under N2. The
`yield of product was typically ~ 80%.
`
`Preparation of dimethyl acetal hydrazido-HA (3)
`
`Preparation of N-(2,2-dimethoxyethyl)-4-(methoxycarbonyl)
`butanamide (1). EDC (4.98 g, 0.026 mol) was added to a mix-
`ture of aminoacetaldehyde dimethyl acetal (2.18 mL, 20
`mmol; Aldrich) and methyl monoester of succinic acid (2.64
`g, 20 mmol; Aldrich) in 75 mL of dichloromethane, and the
`reaction mixture was stirred for 24 h at room temperature.
`The solution was extracted successively with 50 mL each of
`ice-cold solutions of 0.75 M sulfuric acid, 1M NaCl, satu-
`rated sodium bicarbonate, and 1M NaCl. The organic phase
`was collected and dried with sodium sulfate. The solvent
`was evaporated under reduced pressure yielding a syrup
`which showed a single spot on charring upon thin-layer
`chromatography (TLC) in solvent A (Rf 0.75) and solvent B
`(Rf 0.24) (see Characterization: Analytical Methods). The ap-
`parent yield of 1 was 65% and the 1H-NMR spectra in deu-
`terated chloroform showed the following peaks: d 5.70 (bs,
`1H, NH), 4.34 (t, 1H, CH-(OCH3)), 3.67 (s, 3H, COOCH3),
`3.43–3.35 (s and t, 8H, CH3OC and CHCH2NH), 2.38–2.26
`(m, 4H, CH2CO).
`Formation of N-(2,2-dimethoxyethyl)-4-(hydrazido)butanamide
`(2) from 1. Anhydrous hydrazine (248 mL, 7.9 mmol; Aldrich)
`was added to a solution of 1 (1.73 g, 7.9 mmol) in 5 mL of
`anhydrous methanol. The mixture was stirred at room tem-
`perature overnight and the solvent subsequently evaporated
`under reduced pressure, yielding a solid residue. The resi-
`due was dissolved in H2O (6 mL) and extracted three times
`with an equal volume of dichloromethane. The aqueous so-
`lution was evaporated to dryness under reduced pressure
`and then further dried overnight in vacuo. The crystalline
`solid (1.04 g, 82% yield) was homogeneous on TLC in sol-
`vent A (Rf 0.10) when visualized by charring. The 1H-NMR
`spectrum indicated the loss of the ester methoxy group
`when compared to 1.
`Sodium hyaluronate was reacted with acyl hydrazide (2)
`using HOBt and EDC as described above, yielding 3 with an
`apparent degree of modification of ~ 65%. The 1H-NMR
`spectra in deuterated water [Fig. 3(B)] showed peaks at d
`3.28 (s, 6H, CH(OCH3)2), 2.50 (bs, 2H, CO ? CH2), 1.9 (bs, 3H,
`HA-NHCO ? CH3), 1.82 (bs, 2H, CH2
`? CO).
`
`1H-NMR of native and modified HA in D2O at
`Figure 3.
`270 Mhz: (A) native HA, (B) N-(2,2-dimethoxyethyl)-4-
`(hydrazido)butanamide-modified HA (3), and (C) lysine
`methyl ester-modified HA (6). Peaks are assigned as indi-
`cated on the structural formula and detailed in Materials
`and Methods. 1H-NMR results for other HA derivatives are
`provided in Materials and Methods.
`
`Preparation of 2-aminoethyl-HA (5).
`
`Sodium hyaluronate was reacted with ethylenediamine
`dihydrochloride (998 mg; Aldrich) using HOBt and EDC as
`described above. The apparent degree of modification was
`~ 25%: 1H-NMR (D2O) d 3.07 (t, 2H, CO ? NHCH2), 2.65 (bs,
`2H, CH2NH2), 1.9 (bs, 3H, HA-NHCO ? CH3).
`
`Preparation of adipic dihydrazide-HA (4)
`
`Preparation of lysine methyl ester-HA (6).
`
`Sodium hyaluronate was reacted with adipic dihydrazide
`(1.31 g; Aldrich) using HOBt and EDC as described above.
`The apparent degree of modification was ~ 65%: 1H-NMR
`(D2O) d 2.20 (m, 2H, NHNHCO ? CH2), 2.10 (m, 2H,
`CH2NHNH2), 1.35–1.55 (m, 4H, CH2CH2).
`
`Sodium hyaluronate was reacted with lysine methyl ester
`(1.748 g; Sigma, St. Louis, MO) using HOBt and EDC as
`described above. The apparent degree of modification was
`~ 25%: 1H-NMR (D2O) d 3.85 (s, 3H, CO2CH3), 3.0 (t, 2H,
`CH2NH2), 2.1 (bs, 3H, HA-NHCO ? CH3), 2.0–1.83 (dm, 2H,
`
`

`

`156
`
`BULPITT AND AESCHLIMANN
`
`NHCHCH2CH2), 1.81–1.70 (m, 2H, CH2CH2), 1.52–1.43 (m,
`2H, CH2CH2CH2) [Fig. 3(C)].
`
`Preparation of polyaldehyde by oxidation of HA
`
`General procedure of carbodiimide/sulfo-NHS
`reactions with HA
`
`To an aqueous solution of sodium hyaluronate (3 mg/mL)
`was added a 30-fold molar excess of an amine (pKa > 8.5; 7.5
`mmol), i.e., 1,4-diaminobutane. The pH of the reaction mix-
`ture was adjusted to 7.5 with 0.1M NaOH/0.1M HCl. EDC
`(192 mg, 1 mmol) and N-hydroxysulfosuccinimide (sulfo-
`NHS) (217 mg, 1 mmol; Fluka) were dissolved in H2O (1
`mL). After mixing, the pH of the reaction was maintained at
`7.5 by the addition of 0.1M NaOH and the reaction was
`allowed to proceed overnight. The HA derivatives were pu-
`rified and stored as described above.
`
`Preparation of aminoacetaldehyde dimethyl
`acetal-HA (7)
`
`Sodium hyaluronate was reacted with aminoacetaldehyde
`dimethyl acetal (781 mg) using sulfo-NHS and EDC as de-
`scribed above. The apparent degree of modification was
`~ 10%: 1H-NMR (D2O) d 3.30 (s, 6H, CH(OCH3)2), 1.9 (bs, 3H,
`HA-NHCO ? CH3).
`
`Preparation of 4-aminobutyl-HA (8)
`
`Sodium hyaluronate was reacted with 1,4-diaminobutane
`dihydrochloride (1.21 g; Aldrich) using sulfo-NHS and EDC
`as described above. The apparent degree of modification
`was ~ 10%: 1H-NMR (D2O) d 2.87 (m, 4H, CO ? NHCH2,
`CH2NH2), 1.9 (bs, 3H, HA-NHCO ? CH3), 1.55–1.35 (dm, 4H,
`CH2CH2).
`
`Preparation of 6-aminohexyl-HA (9)
`
`Sodium hyaluronate was reacted with 1,6-diaminohexane
`dihydrochloride (1.42 g; Aldrich) using sulfo-NHS and EDC
`as described above. The apparent degree of modification
`was ~ 10%: 1H-NMR (D2O) d 2.9 (m, 4H, CO ? NHCH2,
`CH2NH2), 1.9 (bs, 3H, HA-NHCO ? CH3), 1.6–1.1 (m, 6H,
`CH2CH2CH2).
`
`Sodium hyaluronate (300 mg, 0.75 mmol) was dissolved in
`H2O at a concentration of 20 mg/mL. Sodium periodate (160
`mg, 0.75 mmol; Aldrich) was added and the reaction al-
`lowed to proceed for 2 h at room temperature. Low-
`molecular-weight reaction products were removed by ex-
`haustive dialysis (Spectra/Por RC 2) and the polyaldehyde
`polymer solution was saturated with nitrogen by bubbling
`and stored at 4°C.
`
`Characterization of HA derivatives
`
`Analytical methods
`
`Thin-layer chromatography was done with silica gel 60
`F254 (Merck). The solvent systems used were (a) 4:1 (v/v)
`ethyl acetate:acetone; and (b) 3:1 (v/v) toluene:methanol.
`The 1H-NMR spectra were obtained with a Jeol E-270 instru-
`ment at 270 MHz. For NMR spectroscopy, HA was dis-
`solved in D2O/NaOD, pH 14, at a concentration of 1–2 mg/
`mL. Compound x admixed with HA served as a control for
`HA modified with compound x.
`
`Size distribution
`
`Preparations of HA or chondroitin sulfate (type A from
`bovine trachea, MW ~ 45,000; Sigma) were dissolved in PBS
`at a concentration of 1 mg/mL and 1 mL applied on a Su-
`perose 6 FPLC column (HR 10/30; Pharmacia) equilibrated
`in PBS (5.6 mM Na2HPO4, 1.06 mM KH2PO4, pH 7.4, 154
`mM NaCl). The flow rate was 0.25 mL/min and 0.5-mL
`fractions were collected and the absorbance at 205 nm re-
`corded. Elution profiles of different forms of HA under con-
`ditions minimizing HA–HA interactions (50 mM Na2HPO4,
`pH 11, 500 mM NaCl) were identical. N-Deacetylated HA
`was prepared according to Dahl et al.20 Briefly, high-
`molecular-weight (MW > 106) HA (10 mg, 0.025 mmol) and
`hydrazine sulfate (5 mg, 0.038 mmol) were mixed with an-
`hydrous hydrazine (0.6 mL) in a glass tube. The tube was
`sealed and heated to 100°C for time intervals ranging from 0
`to 180 min. After rapid cooling on ice, 0.75 mL of toluene
`was added and the mixture evaporated to dryness under
`reduced pressure. HA preparations were digested with 100
`U/mL testicular hyaluronidase for 2 h.
`
`Deprotection of HA-acetals to form HA-aldehydes
`
`Formation of HA hydrogels
`
`The acetal modified HA was dissolved in H2O at a con-
`centration of 5–10 mg/mL and 1M HCl was added to give a
`final concentration of 0.025M HCl. The solution was then
`allowed to stand at room temperature for 0.5–1.0 h. The
`solution was neutralized by the addition of 1M NaOH,
`yielding the deprotected HA-aldehyde.
`
`General procedure for crosslinking of
`HA derivatives
`
`Functionalized HA was dissolved by agitation at room
`temperature in PBS, pH 7.4–8.5, at a concentration of up to
`20 mg/mL to study the formation of hydrogels at different
`
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`

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`157
`
`HA concentrations, and at a concentration of 13–15 mg/mL
`for subsequent experiments. The degree of modification of
`the HA derivative was derived from the integration of the
`1H-NMR peaks. After complete dissolution, the HA solution
`was transferred into a 1-mL syringe. When reacting HA with
`low-MW crosslinkers, a slight excess of the compound (1.1
`molar equivalent of functional groups) was used in a second
`1-mL syringe. Polyethylene glycol bis(succinimidyl propio-
`nate) ((SPA)2-PEG) (MW ~ 3400) and succinimide ester of
`carboxymethylated four arm polyethylene glycol ((SC)4-
`PEG) (MW ~ 20,000) were purchased from Shearwater Poly-
`mers (Huntsville, AL), 3,38-dithiobis (sulfosuccinimidyl pro-
`pionate) (DTSSP) from Pierce Chemical Co., and glutaralde-
`hyde from Fluka. The solution was freshly prepared in PBS
`at 1/10 of the HA volume. The syringes were connected
`while paying special attention to exclude air; the contents
`were rapidly mixed with 20 passages and allowed to gel.
`When reacting HA molecules with different functionalities,
`0.5–1 equivalent of HA-aldehyde was mixed with 1 equiva-
`lent of HA-hydrazide, depending on the degree of modifi-
`cation of the HA derivatives. At room temperature, gelation
`occurred within 30 s to several minutes, depending on the
`formulation, and the gel properties did not significantly
`change after approximately 5 min.
`
`In vitro and in vivo testing of HA hydrogels
`
`Hyaluronidase digestion
`
`Hyaluronic acid hydrogels were formed as detailed above
`in 1-mL syringes. The syringes containing the crosslinked
`gels were incubated at 37°C for 1 h to ensure the crosslinking
`reaction was complete, after which identical ~ 100-mL cylin-
`drical gels were formed by cutting the syringes with a razor
`blade and extruding the gels. The gels were incubated at
`37°C with different concentrations of bovine testicular hyal-
`uronidase (Sigma), 50–5000 U/mL, in 400 mL of 30 mM citric
`acid, 150 mM Na2HPO4, pH 6.3, containing 150 mM NaCl,
`for the indicated time (0–48 h). Degradation of the gels was
`determined from the release of glucuronic acid into the su-
`pernatant as measured by the carbazole assay.26
`
`Rat subcutaneous implantation
`
`Biomaterials were implanted in 4- to 5-week-old male
`Sprague-Dawley rats. Briefly, a small vertical incision was
`made on either side of the xiphoid cartilage of the sternum,
`and the skin undermined with a blunt instrument to sepa-
`rate the skin from the underlying tissue. Biomaterial discs
`(10 mm in diameter, 3 mm width) were placed into these
`pockets and the skin incisions closed with sutures. For each
`test group, four to six biomaterial specimens were im-
`planted. Animal experiments were performed according to
`NIH Guidelines for the Care and Use of Laboratory Animals
`(NIH Publ. No. 85-23, Rev. 1985).
`Hyaluronic acid hydrogel discs were prepared by
`crosslinking HA derivatives (12–14 mg/mL) in PBS in 3-mL
`syringes as described above. The HA solution was supple-
`
`mented with cell adhesion molecules such as collagen type I
`fibrils (1 mg/mL) or fibronectin (500 mg/mL) prior to
`crosslinking when desired to promote cell infiltration. Col-
`lagen fibrils were prepared by polymerization from dilute
`solution (2–3 mg/mL) of acid-solubilized intact bovine col-
`lagen type I (retaining telopeptides; Organogenesis, Canton,
`MA) in PBS and harvested by centrifugation, following stan-
`dard protocols.27 For induction of chondro-osseous differ-
`entiation, different growth factors including bone morpho-
`genetic protein-2 (BMP-2) (200 mg/mL; Genetics Institute,
`Cambridge, MA), insulin-like growth factor-1 (IGF-1) (500
`ng/mL; Celtrix Pharmaceuticals, Santa Clara, CA), and
`transforming growth factor-b2 (TGF-b2) (50 ng/mL; Celtrix)
`were mixed with the HA solution just prior to crosslinking.
`For inhibition of vascularization, 10 mg/mL suramin
`(Sigma) was added.
`At the indicated time postoperatively, the animals were
`euthanized, and the implants were exposed through a dorsal
`midline skin incision and excised with the attached sur-
`rounding tissue. The tissue was fixed in 10% formalin in
`PBS, dehydrated through a graded ethanol series, and em-
`bedded in paraffin. Five-micrometer sections were cut and
`stained with hematoxylin/eosin and Safranin-O/Fast green.
`
`RESULTS AND DISCUSSION
`
`We focused on the production of hydrogels from
`HA, since HA matches several of the desired proper-
`ties for a biomaterial for delivery of bioactive agents
`such as cells, growth factors, cytokines, and drugs for
`tissue repair. It is biodegradable, provides an excellent
`substratum for cell migration, and, most important,
`has proven its biocompatibility in various forms in
`clinical practice (for discussion, see Balazs and Laur-
`ent.8) The relatively simple repetitive structure of HA
`allows also for specific modification and introduction
`of a large number of functional groups for subsequent
`crosslinking (Fig. 1).
`
`Preparation of HA derivatives
`
`Initial studies
`
`It is well known that sugars can be oxidized using
`periodate,28 and initially, we followed standard pro-
`cedures to generate polyaldehydes from HA by per-
`iodate oxidation. Periodate treatment oxidizes the
`proximal hydroxyl groups (at C2 and C3 carbons of
`glucuronic acid moiety) to aldehydes, thereby opening
`the sugar ring to form a linear chain. While periodate
`oxidation allows for the formation of a large number
`of functional groups, the disadvantage is the loss of
`the native backbone structure. Consequently, the gen-
`erated derivative is presumably not recognized as HA
`by cells. Indeed, hydrogels formed by using periodate-
`oxidized HA as a crosslinker, e.g., in combination with
`the HA-amine derivatives described below, showed
`very limited tissue transformation and poor cellular
`infiltration in the rat ectopic bone formation assay (see
`
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`158
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`BULPITT AND AESCHLIMANN
`
`below) (Table I). Clearly, periodate oxidation of HA
`was not suitable and our goal had to be to generate an
`activated form of HA that differs minimally from na-
`tive HA to conserve its unique physicochemical prop-
`erties. Also, a minimal change affecting only a rela-
`tively small number of dissaccharide units would pre-
`sumably not alter its property to serve as a cell
`substratum, since short HA oligosaccharides are rec-
`ognized by HA receptors.6 Since deacetylation of the
`N-acetylglucosamine moiety leads to concurrent deg-
`radation of the HA chain19,20 [Fig. 4(A)], our efforts
`focused on the carboxylate group of the glucuronic
`acid moiety (Fig. 1). Initially, attempts were made to
`generate an aldehyde derivative by reduction of the
`carboxyl groups on the glucuronic acid residues using
`9-borabicyclo-3,3-nonane, a method that allows direct
`conversion of the carboxylic acid into an aldehyde.29
`However, this avenue of investigation was unsuccess-
`ful, and we decided to introduce an aldehyde onto HA
`by means of a side chain using carbodiimide-mediated
`coupling.
`
`Introduction of a (functionalized) side chain
`onto HA
`
`Direct carbodiimide-mediated coupling of amines
`to the carboxyl group of HA in an aqueous environ-
`ment, e.g., with 1-ethyl-3-[3-dimethylaminopropyl]
`carbodiimide (EDC), did not yield the predicted prod-
`uct since the O-acyl isourea that is formed as a reactive
`intermediate rearranges rapidly to a stable N-acyl urea
`(Fig. 2). This results in predominant coupling of EDC
`(>99%) as indicated by the 1H-NMR spectrum which
`
`showed the characteristic sharp singlet at d = 2.08 cor-
`responding to the N-methyl protons and the methy-
`lene protons of the N-ethyl methyl group at d = 1.0,
`which is consistent with previous reports.22,23 We
`found that the coupling of primary amines to HA is
`possible via the formation of a more hydrolysis resis-
`tant and non-rearrangable active ester intermediate
`(Fig. 2). We formed active esters of HA with 1-hy-
`droxybenzotriazole (HOBt) or N-hydroxysulfosuccin-
`imide (Sulfo-NHS) using the water-soluble carbodi-
`imide EDC for coupling. Nucleophilic addition to the
`ester formed from HOBt requires the amine to be pre-
`sented in an unprotonated form at acidic pH (about
`5.5–7.0). Only a limited number of amines including
`hydrazides and “activated” amines, e.g., ethylene di-
`amine, have pKa values in a suitable range and are
`consequently relatively unprotonated and reactive
`with the ester-intermediate formed with HOBt (Fig. 2:
`3–6). Simple primary amines, e.g., putrescine, which
`have typically pKa values > 9 do not form significant
`amounts of adduct under acidic coupling conditions.
`The Sulfo-NHS-derived intermediate allows for the
`coupling reaction to be carried out at neutral or
`slightly basic pH (about 7.0–8.5) and consequently
`yields products by reaction with simple primary
`amines (Fig. 2: 7–9).
`
`Preparation of HA-aldehyde derivatives
`
`A heterobifunctional side chain, N-(2,2-dimeth-
`oxyethyl)-4-(hydrazido) butanamide (2), containing a
`protected aldehyde in the form of an acetal and an acyl
`hydrazide for coupling was synthesized using stan-
`
`TABLE I
`Evaluation of Biological Properties of Different HA Hydrogels in Rat Subcutaneous Implantation Model
`
`Crosslinker
`
`Granulomatous
`Inflammation*
`
`Cell Infiltration†
`
`Chondro-osseous
`Differentiation‡
`
`Group
`
`1
`2
`3
`4
`5
`6
`7
`
`8
`
`HA Derivative
`~ 25% AD-HA
`~ 25% AD-HA
`~ 25% AD-HA
`~ 65% AD-HA
`~ 25% AD-HA
`~ 25% AD-HA
`~ 25% AD-HA
`~ 25% AD-HA
`
`+++
`+
`+
`+
`+++
`+
`++
`
`+/++
`
`−/+
`−
`++
`+
`+
`+/++
`+++
`
`++
`
`−
`−
`+++
`+/++
`−
`+/++
`+
`
`++
`
`Glutaraldehyde
`DTSSP
`(SPA)2-PEG
`(SPA)2-PEG
`Periodate-oxidized dextran
`Periodate-oxidized HA
`~ 10% aminoacetaldehyde
`dimethyl acetal-modified HA
`~ 65% N-(2,2-dimethoxyethyl)-
`4-(hydrazido)butanamide-
`modified HA
`~ 25% LME-HA
`9
`(SPA)2-PEG
`+/++
`+++
`++
`HA hydrogels were formed by crosslinking 12 mg/mL of the HA-amine derivatives (adipic dihydrazide-[AD-HA] or lysine
`methylester-[LME-HA] modified HA) with a slight excess of crosslinker (groups 1–4 and 9) or by mixing polysaccharide
`derivatives with different modifications such as to yield the same final polysaccharide concentration (groups 5–8), as de-
`scribed in Materials and Methods. Hydrogels were supplemented with prefibrillized collagen type I and BMP-2 to stimulate
`endochondral bone formation (see Table II).
`*+ = mild inflammation with mononuclear cells; ++ = moderate with occasional foreign-body giant cells; +++ = severe with
`abundant presence of foreign-body giant cells.
`†+ = <10% of implant infiltrated; ++ = >30%; +++ = >80%.
`‡+ = occasional foci of cartilage/bone; ++ = thin layer surrounding implant; +++ = >30% of implant replaced.
`
`

`

`IN SITU POLYMERIZABLE HYALURONIC ACID HYDROGEL MATERIALS
`
`159
`
`Figure 4. Size distribution of derivatized HA. HA preparations were separated on a Superose 6 FPLC column in PBS and
`the absorbance at 205 nm was recorded. (A) HA (MW > 106) was N-deacetylated by hydrazinolyses for the indicated time
`(0–180 min). (B) HA was derivatized with lysine methyl ester (6) or putrescine (8) via a benzotriazole or sulfosuccinimide ester
`intermediate, respectively. (C) HA and HA derivatives were digested with 100 U/mL of testicular hyaluronidase for 2 h (see
`next page). The void volume (v0) and total volume (vT) of the column are indicated by arrows. The elution of chondroitin
`sulfate (CS) with a molecular mass of ~ 45 kDa is indicated in (A).
`
`dard chemistry as detailed in Materials and Methods.
`The side chain was coupled to HA as outlined in Fig-
`ure 2 (3). High-molecular-weight HA (MW > 106) was
`dissolved in water, and a 20-fold molar excess of the
`
`side chain was added to ensure efficient coupling.
`Next, a fourfold molar excess of HOBt was added and
`the pH adjusted to 6.8. Finally, a fourfold molar excess
`of EDC was added to the reaction mixture, which re-
`
`

`

`160
`
`BULPITT AND AESCHLIMANN
`
`sulted in a slowly progressing decrease in pH. The pH
`was maintained at 6.8 for at least 4 h by the addition of
`0.1M NaOH and the reaction was allowed to proceed
`overnight. The progressing slight decrease in pH pre-
`sumably reflects the consumption of the basic primary
`amine over time since the reaction does not result in a
`net change in protons. This is in sharp contrast to the
`rapid pH increase observed when HA is reacted with
`EDC alone, which has been attributed to the net con-
`sumption of a proton,23 and suggests that the reaction
`proceeds through the active ester intermediate.
`A side chain with protected aldehyde functionality
`on one end and a primary amine for coupling on the
`other end, aminoacetaldehyde dimethyl acetal, was in-
`corporated into HA in a similar manner (Fig. 2: 7).
`HOBt was replaced with a fourfold molar excess of
`Sulfo-NHS and the pH was adjusted to 7.5. Upon ad-
`dition of EDC, a similar drop in pH was observed. The
`pH was maintained at 7.5 and the reaction was al-
`lowed to proceed overnight.
`The HA derivatives were purified by exhaustive di-
`alysis against water followed by repeated ethanol pre-
`cipitation to remove unreacted amine and other small
`reaction products. 1H-NMR confirmed that the modi-
`fication was successful, with the characteristic appear-
`ance of the acetal protons at d = 3.30 [Fig. 3(B)]. The
`HA-acetal derivatives were easily activated to the re-
`active aldehydes by mild acid treatment, and when
`mixed with dihydrazides or dihydrazide-modified
`HA (see below) produced hydrogels, further confirm-
`ing that successful modification had been achieved.
`
`Preparation of HA-amine derivatives
`
`Introduction of homobifunctional amines, i.e., eth-
`ylene diamine, lysine methyl ester, histidine methyl
`ester, and adipic, succinic, or suberic dihydrazide,
`which contain at least one amino group with a pKa <
`8.0, onto HA was conducted in a similar manner using
`HOBt and EDC (Fig. 2: 4–6). The degree of modifica-
`tion could be controlled between ~ 10% and 25% for
`amines and ~ 10% and 70% for hydrazides by adjusting
`the molar equivalency ratio of HA:EDC from 1:0.5 to
`1:4. The HA derivatives were purified as described
`above and analyzed by 1H-NMR. The spectra revealed
`that modification was successful. For example, the HA
`modified with lysine methyl ester showed the charac-
`teristic singlet at d = 3.85 corresponding to the methyl
`protons of the methyl ester group and the signal at d =
`3.0 corresponded to the methylene protons adjacent to
`the amino group [Fig. 3(C)]. The remaining methylene
`protons appeared between d = 1.4 and 2.0, and
`showed the expected characteristic splitting patterns.
`Homobifunctional amines having amino groups with
`pKa > 9.0, i.e., 1,4-diaminobutane (putrescine) and 1,6-
`diaminohexane, were coupled to HA using Sulfo-NHS
`and EDC (Fig. 2: 8,9). In this case, the degree of modi-
`fication with a fourfold excess of EDC was ~ 10% as
`assessed by 1H-NMR. The ability of these HA deriva-
`tives (4–6,8,9) to form hydrogels with amino group
`specific crosslinkers such as aldehydes and