New Gelatin-Based Hydrogels via Enzymatic Networking

Department of Chemistry, University “La Sapienza”, Rome, Italy ..... Giuseppe Tronci , Axel Thomas Neffe , Benjamin Franklin Pierce , Andreas Lend...
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Biomacromolecules 2002, 3, 1384-1391

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New Gelatin-Based Hydrogels via Enzymatic Networking Vittorio Crescenzi,* Andrea Francescangeli, and Anna Taglienti Department of Chemistry, University “La Sapienza”, Rome, Italy Received August 30, 2002

New types of hydrogels have been obtained starting from high bloom purified gelatin A, alone or in mixtures with hyaluronan and with a hyaluronan derivative bearing primary amino groups, by transglutaminasecatalyzed cross-linking. The reticulation process, carried out adopting two different temperature protocols, and the ensuing materials have been characterized in terms of rheologically estimated gel times, equilibrium swelling in water and in phosphate buffer solution (PBS), and rigidity modulus. Main structural and conformational factors governing the physicochemical properties and the possible application of the new hydrogels are discussed. 1. Introduction A variety of chemical hydrogels1-3 have been synthesized starting from a number of different biopolymers, mainly polypeptides (e.g., collagens and gelatins4) and polysaccharides (e.g., hyaluronic acid,5-7 chitosan8,9). However, despite the biocompatible nature of the starting products, the chemical reticulation processes normally employed may jeopardize the use of the ensuing materials, even if thoroughly purified, because of the unpredictable biocompatibility level of the chains’ bridging moieties. Our studies aim at preparing and characterizing new hydrogels (and at exploring their application in the medical sector) based on selected natural and synthetic polypeptides and on hyaluronan or hyaluronan derivatives or both using as cross-linking agent exclusively an enzyme, namely, a tranglutaminase of microbial origin10 (MTGase). As is known, transglutaminases (amine γ-glutaminyl transferase, EC 2.3.2.13) catalyze the cross-linking of proteins promoting the formation of isopeptide bonds between the γ-carbonyl group of a glutamine residue and the -amino group of a lysine residue.11 The transglutaminase produced by a variant of StreptoVerticillium mobaraense, of which the catalytic activity is Ca(II) ion-independent, differing from transglutaminases of human or animal origin, is highly specific toward the acyl donor. On the other hand, besides lysine residues as acyl acceptors, primary alkylamines can be used as substrates, which allows the selective alkylation of proteins via their accessible glutamine residues.12 To our knowledge, MTGase has been used mainly for food applications,13 for the immobilization of proteins in caseine gels,14,15 or on ion-exchange resins, etc. We wish to report here our results obtained employing MGTase to cross-link, in aqueous media at two different temperatures, purified high bloom gelatin A alone or in combination with hyaluronan16 (HA, Figure 1) and with a * To whom correspondence should be addressed. E-mail: vittorio. [email protected].

HA derivative bearing lysil substituents. The presence of HA in a gelatin gel makes the network distinctly more hydrophilic and in particular “bioactive”, considering the ability of hyaluronan to promote cell regeneration.2 Such mixed gels should provide very good materials to replace fleshy tissues, such as the nucleus pulposus; to this end, the possibility to modulate the networks’ mechanical properties and swelling capacity and to perform the enzymatic cross-linking in situ are fundamental factors. Some basic features of physical and chemical gelation of gelatins A and B useful for a better insight into the properties of mixed gels of our main concern are also briefly pointed out with the aid of a few relevant data. 2. Materials and Methods 2.1. Materials. A hyaluronic acid (HA) sample, a Fidia Advanced Biopolymers (FAB Srl, Abano Terme, Padua, Italy) product, Mη ) 145 kDa, has been used throughout. Gelatin samples (A1, A2, and A3 acid extracted from porcine skin with bloom number equal to 75-100, 175, and 300, respectively; B1 and B2 alkali extracted from bovine skin with bloom number equal to 75 and 225, respectively) were products of Sigma, Milano, Italy. The amino acid composition of samples considered is given in Table 1. Ca2+independent transglutaminase (MTGase) derived from the microorganism StreptoVerticillium was kindly supplied by Ajinomoto Co. Inc. (Japan). According to information from the supplier, the enzyme concentration in the preparation accounts for 10% of the total material, the remainder being maltodextrin (90%). All other chemicals were reagent grade and have been used without further purification. 2.2. Synthesis of Hyaluronic Acid Derivative HA-GK. To prepare mixed chemical gels based on gelatin and hyaluronan, the polysaccharide has been functionalized with appropriate amounts of H-Gly-Lys dipeptide17-21 (GK). In practice, 500 mg of HA was dissolved in 20 mL of MES buffer (50 mM, pH ) 4.0), and about 1 g of glycine-lysine hydrochloride was added (a large excess to minimize the

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New Gelatin-Based Hydrogels Table 1. Amino Acid Composition of Gelatinsa gelatin amino acid

A1

A2

A3

B1

B2

N + D + P-OH asparagine + 5.0 4.8 4.7 4.7 4.7 aspartic acid + hydroxyproline T threonine 1.1 1.1 1.1 1.1 1.1 S serine 3.5 3.3 3.2 3.3 3.4 Q+E glutamine + 8.4 8.3 8.4 8.5 8.4 glutamic acid P proline 10.3 10.2 10.4 10.1 9.8 G glycine 43.3 42.8 42.5 42.4 41.4 A alanine 12.2 13.5 13.6 14.2 15.2 V valine 2.1 2.2 2.3 2.1 2.1 M methionine 0.7 0.6 0.6 0.5 0.4 I isoleucine 1 0.9 0.9 1.1 1.1 L leucine 2.1 2.2 2.2 2.2 2.2 Y tyrosine 0.3 0.3 0.3 0.2 0.2 F phenylalanine 1.1 1.2 1.2 1.2 1.2 K-OH hydroxylysine 0.6 0.6 0.4 0.5 0.5 H histidine 0.7 0.5 0.5 0.5 0.4 K lysine 2.9 2.8 2.6 2.6 2.7 R arginine 4.4 4.6 4.7 4.5 4.6 a Hydrolysis has been carried out with HCl (6 N) at 110 °C for 24 h; the values indicate the number of the amino acid per 100 residues.

probability of a concomitant cross-linking process). Three hundred sixty milligrams of EDC‚HCl [N-(3-dimethylaminopropyl)-N′-ethylcarbodimide hydrochloride)] and 220 mg of NHS (N-hydroxysuccinimide) were then added, keeping the following molar ratios: [EDC] ) 1.5 [COOH]

[NHS] )1 [EDC]

The mixture has been stirred for 24 h at room temperature and then dialyzed against a 0.1 M Na2CO3 aqueous solution overnight (to hydrolyze ester bonds) and then extensively against water and finally freeze-dried. The reaction described above does not allow us to arrive at a derivative of univocal structure (Figure 1) but does allow the insertion of a short alkyl spacer between the polysaccharide backbone and a free, primary amino functionality (when the attachment of the dipeptide onto the HA chains takes place via the glycyl residue, of course). According to NMR data, the molar ratio of bound dipeptide to HA repeating units is approximately 0.10. Viscosity measurements suggest that the derivatized sample has an average molecular weight only slightly less than that of the starting HA (probably because of limited chain degradation during the dialysis against sodium carbonate). 2.3. Gelatin A3 Purification. Twenty grams of A3 commercial gelatin was suspended in 100 mL of water (20% w/v), and the solution was kept under stirring for 30 min. The mixture was heated to 60° C for 10 min, then cooled to room temperature, and let to stand still overnight. The gel obtained was dialyzed (at 4° C) against repeated changes of distilled water for several days to eliminate salts (dialysis water finally reaches the nominal conductivity of distilled water). The gel was put in 5 L of distilled water, stirred, and heated to 60° C to disaggregate the physical network. The final solution was filtered on sintered glass and then

Figure 1. Structure (a) of hyaluronic acid sodium salt repeating unit and (b,c) of hyaluronic acid-glycine-lysine (HA-GK) representative repeating unit (with a degree of derivatization, for b + c, equal to 10%).

freeze-dried. The elimination of salts was checked by measuring the conductivity of a 0.2% w/v solution of both commercial and purified gelatin (47.3 and 17.0 µS, respectively). 2.4. Viscosity Measurements. The viscosity (25 °C) of dilute HA, HA-GK (T ) 25 °C, 0.2 M NaCl aqueous solutions), and purified gelatin A3 (T ) 50 °C, PBS, pH ) 7.0) has been measured using a Schotte-Gerate automatic dilution viscometer (AVS). Intrinsic viscosity data have been calculated by a double extrapolation of the relevant Huggins-Kraemer plots. 2.5. Preparation of Physical and Chemical Gels using Gelatins A and B. To check the suitability of different A and B gelatin samples for the preparation of physical gels and, in particular, in the synthesis of chemical networks, commercial gelatins A and B (see point 2.1 above) were allowed to swell in 8 mL of PBS (pH ) 7.0) for 30 min at room temperature to prepare solutions of 3%, 4.5%, 6%, 7.5%, 9%, and 10.5% w/v final polymer concentration for each sample considered, assuming for the commercial samples a purity of 75%. The polymer was completely

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Table 2. Formation of Physical or Chemical Gelatin Gels or Both A1 (75-100)a A2 (∼75)a A3 (∼300)a B1 (∼75)a B2 (∼225)a

CGb

Pc

Cd

P

C

P

C

P

C

P

C

3 4.5 6 7.5 9 10.5

-

-

x x x

x x x

x x x x x

x x x x x x

x x x x x x

-

x x x x x x

-

a The value in the parentheses indicates the “bloom number”: 50125 w MW ) 20-25 kDa; 125-225 w MW ) 40-50 kDa; 225-325 w MW ) 50-100 kDa. b Gelatin concentration expressed as % w/v. c P ) physical gel obtained after 5 h at 50 °C and overnight at room temperature. d C ) chemical gel (via MTGase) obtained after 5 h at 50 °C and overnight at room temperature.

dissolved at 60 °C, and the solutions subsequently were cooled to the reaction temperature (50 °C). MTGase preparation was added (200, 300, 400, 500, 600, and 700 µL, respectively, of a 1% w/v enzyme solution in PBS, to provide a constant gelatin/enzyme ratio equal to 150 w/w), and the mixture was immediately made up to 10 mL with PBS to obtain the desired final concentrations. After incubation at 50 °C for 5 h, the system was cooled to room temperature and let stand overnight. All gels were subjected to extensive dialysis against distilled water. To obtain gelatin physical networks, the same procedure was carried out except for the addition of the enzyme. The results of the experiments are schematically summarized in Table 2. 2.6. Synthesis of Gels Starting from A3, A3/HA, and A3/HA-GK Systems. Chemical gels of A3 alone for the swelling measurements have been prepared using the gelatin solutions specified at point 2.4; gel formation has been carried out for 8 h at 37 °C or for 1 h at 50 °C and 7 h at 37 °C. These two temperature protocols will be indicated as protocols a and b, respectively, in what follows. To test the influence of the presence of HA and HA-GK, 450 mg of purified A3 gelatin was allowed to swell in 8 mL of PBS (pH ) 7.0) for 30 min at room temperature. The polymer was completely dissolved at 60 °C, and the solutions were subsequently cooled to 50 °C. MTGase preparation was added (300 µL of a solution 1% w/v in PBS), and the mixture was immediately made up to 10 mL with PBS to obtain the final concentration. The procedure was repeated in the presence of HA and of HA-GK (5%, 10%, and 20% w/w with respect to gelatin A3) for the two protocols a and b. 2.7. Thermal Gel-Sol Transition of Physical Gel. Gelsol transition of the A3 gelatin (4.5% w/v) was monitored by measuring the specific optical rotation ([R]D) as function of temperature. Experiments were performed with a PerkinElmer polarimeter 241 using a 1 cm quartz cell. 2.8. UV Measurements. To study the gelatin-HA coacervation process, absorbance at 800 nm was measured for the system A3/HA (gelatin and HA concentration equal to 4.5% and 0.45% w/v, respectively) at different pH values in water and in 0.2 M NaCl by 0.01 M HCl addition. Absorbance data were collected with a HP 8452A diode array spectrophotometer using a 1 cm quartz cell. 2.9. Swelling Measurements. The equilibrium water swelling (SW) of the hydrogels is defined as the ratio between

the weight of the networks in the swollen state (WS) after 10 days dialysis against repeated changes of distilled water (at 25 °C) and in the freeze-dried state (WD): SW )

WS WD

Swelling of networks in PBS at 25 and at 37 °C has been determined knowing the hydrogel weight in water (WS) and measuring its weight (WPBS) after 2 days of dialysis against 25° 37° ) and 37 °C (WPBS ): a PBS solution (pH ) 7.0) at 25 (WPBS 25° SPBS )

25° WPBS

W25° S

S25° W

37° SPBS )

37° WPBS

W25° S

S25° W

2.10. Rheological Measurements. A Bohlin CS rheometer was used to follow the gelation of the aqueous systems A3, A3/HA, and A3/HA-GK (polysaccharide/gelatin ratio equal to 10%) and to characterize the gels produced in terms of “equilibrium” rigidity modulus.22 The geometry adopted consisted of coaxial cylinders (C14). The solution containing the enzyme was transferred to the rheometer at 50 °C, and the temperature was brought to the final value. The measurements were taken following either temperature protocol a or temperature protocol b. To prevent evaporation, samples were covered with silicone oil. Gels were subjected to oscillation at a frequency of 0.1 Hz; the amplitude of deformation was chosen at 0.2% strain to remain within the linearity limits of viscoelasticity. A frequency sweep was generally performed after 24 h from 0.01 to 1 Hz. 3. Results and Discussion 3.1. Gel-Forming Ability. Let us consider the physical and chemical (enzymatically catalyzed) gel formation capacity of the different gelatin samples employed. Relevant data are schematically reported in Table 2 for different gelatin concentrations working under standard experimental conditions. Clearly, gelatin A3 with the highest bloom number (and viscosity average molecular weight around 90 kDa) stems out as a superior gelling agent. As expected, gelatin samples B1 and B2 cannot undergo enzymatically catalized chemical gelation inasmuch as they are devoid of glutamine residues (which have been hydrolyzed during the alkaline extraction). Gelatin sample A1 is the worst gelling agent for our working conditions; in fact, it does not form either physical or chemical gels up to a concentration of 10.5% w/v. The average molecular weight of such a sample is estimated to be around 20 kDa. Evidently, the chains are too short to be able to build up sufficiently long helical sections23 (physical gelation) or to undergo sufficiently extensive chemical bridging, according to the reaction scheme presented in Figure 2, to give rise to a polymeric network at the concentrations studied. In addition, it has to be considered that gelatins A have an average content of enzymatically cross-linkable groups, that is, of lysine and glutamine residues, lower than about 3% (See Table 1, Materials and Methods). 3.2. Gel-Sol Transition of Gelatin A3 Physical Gel. It is known that the sol f gel transition for a physical aqueous

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Figure 2. Chemical reticulation promoted by transglutaminase.

Figure 3. Physical gelation of gelatin.

gelatin gel involves formation of triple helical sequences connected in an essentially random fashion by peptide sequences in disordered conformation23 (Figure 3); the transition can be studied by measuring optical rotation as a function of temperature because the gel phase happens to have a chirality due to local conformational order greater than the sol phase. In Figure 4, it is possible to observe how [R]D (specific optical activity at the sodium D line) rises in absolute value as the temperature decreases; below 40 °C, the gel phase begins to form with a typical hysteresis in the cooling-induced gelation process.24 3.3. Swelling in Water of A3, A3/HA, and A3/HA-GK Chemical Gels. The swelling data for gelatin A3 chemical gels in water (Figure 5) show that the Sw values, all relatively small, decrease with increasing polypeptide concentration,

Figure 4. Specific optical rotation as a function of the temperature for purified gelatin A3 (4.5% w/v in PBS): (9) cooling; (2) heating.

as expected, and that the swelling capacity is systematically higher for samples synthesized at 37 °C, a temperature for which enzymatic activity, and hence network connectivity, is obviously less than for the other temperature protocol employed (1 h at 50 °C and 7 h at 37 °C). Water swelling for A3/HA gels (prepared according to either temperature protocol a or b) increases markedly with HA content (Figure 6). Evidently, hyaluronan chains entrapped in the gelatin networks act as efficient water

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Figure 5. Swelling dependence on gelatin concentration for A3 gels, prepared using the temperature protocols a (A3a) and b (A3b): (O) 25° Sw for A3a; (9) Sw for A3b; (2) S25° PBS for A3a; ([) SPBS for A3b; (0) 37° S37° for A3a; (b) S for A3b. PBS PBS

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Figure 7. Swelling vs ionic strength of the different hydrogels: ([) A3a; (2) A3b; (O) (A3/HA)a; (4) (A3/HA)b; (3) (A3/HA-GK)a; (0) (A3/ HA-GK)b. Gelatin concentration equals 4.5% w/v; HA/gelatin ratio equals 0.1 w/w; the letter following the gel type indicates the temperature protocol used.

Figure 8. Absorbance at 800 nm for the A3/HA aqueous system (gelatin and HA concentration equal to 4.5% and 0.45% w/v, respectively) in (b) water and in (2) NaCl 0.2 M.

Figure 6. Swelling data for A3, A3/HA, and A3/HA-GK with different polysaccharide/gelatin ratios (5%, 10%, and 20% w/w) prepared using the temperature protocols (A) a and (B) b: (black bars) Sw; (white 37° bars) S25° PBS; (gray bars) SPBS.

attractants. The effect is less marked in the case of the A3/ HA-GK gels very likely because the HA derivative, though hydrophilic, brings about a modest increase in average degree of cross-linking and hence reduces swelling slightly. 3.4. Swelling in PBS. Once the water equilibrated gels considered above (section 3.3) are brought in contact with an excess of PBS aqueous buffer (pH 7 and ionic strength ca. 0.2) a substantial expansion takes place (except for the A3 gels, see Figure 5) and water uptake reaches values more than twice those in water in the case of the A3/HA (20%)

gels (Figure 6). This phenomenon is, in our opinion, the result of a combined pH-ionic strenght effect. When a polyelectrolyte-based hydrogel is put in a buffer solution, small ions (such as Na+) penetrating inside the network shield the fixed charges on the macromolecular structure leading to a reduction in electrostatic repulsive interactions; the elastic energy may then prevail, and the network shrinks with a swelling decrease (Figure 7). Simultaneously, in our mixed gels, attractive interactions between oppositely charged macroions, HA charged negatively and gelatin charged positively, lead to local aggregate formation with an overall shrinkage of the gel samples. Upon invasion of buffer ions, such aggregates are easily dissolved, as illustrated in Figure 8 (in which the absorbance at 800 nm, only due to light scattering by aggregates, is reported) clearly demonstrating that, for the polymer concentrations employed in the preparation of the gels, HA-gelatin coacervation in water takes place at pH = 4.5 (while even at slightly different pH values the sol state prevails), and that the ionic strength of PBS (simulated with 0.2 M NaCl) completely abolishes coacervation.25 These experimental data (Figures 7 and 8) show

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Figure 9. Sol-gel transition for (A) A3 and (B) A3/HA gel (gelatin concentration equal to 4.5% w/v; HA/gelatin ratio equal to 0.1 w/w) prepared using temperature protocol b: (9) G′; (gray circles) G′′; (4) temperature.

that at pH about 5.5 in the presence of salt (I ≈ 0.2 M) we abolish coacervation but the swelling of the networks decreases; in PBS (pH 7, I ≈ 0.2 M), we also break the aggregates gelatin-HA but the swelling increases because the two polymer chains are more densely charged (HA is almost completely ionized, and the gelatin is near its isoelectric point, pH 9). We have also studied the effect of temperature on the swelling perfomed in PBS; in particular, at 37 °C (human body temperature), the swelling for A3, A3/HA, and A3/ HA-GK is lower than at 25 °C, probably because of hydrophobic interactions between chain portions bearing alkyl residues26 or because of an increase of entropic barriers to chain stretching (rubber theory elasticity).27 3.5. Rheology Data. The aim of the rheological measurements (see Material and Methods) has been that of characterizing the course of the enzymatic cross-linking process of the A3, A3/HA, and A3/HA-GK systems using the two temperature protocols, a and b, in terms of “gel points” (i.e., the time at which G′ ) G′′) and plateau G′ values (Figure 9). The latter are the values of the elasticity modulus for each given gel when the cross-linking reaction has attained an apparent equilibrium or steady-state. Probably, in this state, the enzyme is inactivated by being tightly trapped inside the polymeric network or processes of cross-link wastage such as cyclization and steric inhibition become important or both.

Table 3. Rheological and Swelling Data for A3, A3/HA, and A3/HA-GK “Enzymatic” Gels systema A3 A3/HA A3/HA-GK

temperature protocolb

gel point (min)

G′ (Pa)

Smax (PBS)

a b a b a b

67 25 67 42 67 25

110 70 150 65 120 150

∼18 ∼15 ∼45 ∼70 ∼60 ∼60

a For the systems A3/HA and A3/HA-GK, polysaccharide/gelatin ) 0.1 w/w; gelatin/enzyme ) 150 w/w. b Temperature protocol a ) 37 °C (8 h); temperature protocol b ) 50 °C (1 h) and 37 °C (7 h).

The results indicate that for all systems studied the gel points pertain to shorter times when the cross-linking is carried out using protocol b (Table 3): in fact, 50 °C corresponds to the maximum catalytic performance temperature for MTGase. In addition, for A3 and A3/HA systems, the highest G′ plateau value is reached using protocol a. A likely explanation is that a physical-chemical gel is formed, which would exhibit a relatively high conformational order and therefore a higher rigidity. The opposite happens for the A3/HA-GK system for which G′ attains under protocol b the highest observed value; in this case, participation of the lysyl residues of HA-GK in the reticulation process, characterized by a relatively short gelation time, would increase networking and thus the

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Figure 10. Stress sweep of (A) A3 and (B) A3/HA systems reported in Figure 9: (0) τ ) 1 Hz; (4) τ ) 0.1 Hz; (]) τ ) 0.01 Hz; (O) G′ ) 1 Hz; (3) G′ ) 0.1 Hz; (+) G′ ) 0.01 Hz.

product’s rigidity. Representative results of stress-sweep experiments illustrated in Figure 10 show that the G′ values measured and discussed here can be considered reliable ones. In fact, the plots indicate the absolute independence of G′ and stress with respect to the applied frequencies near the working frequency used in oscillation experiments (0.1 Hz). Moreover, for any given frequency, we verify the theoretical relationships between G′ and strain (constant function) and between stress and strain (linear function). 4. Concluding Remarks With a polymer such as gelatin, one can, at least in principle, arrive at different types of hydrogels. In fact, starting from a physical gel, mobile chain sections of the swollen network can be reacted with a cross-linking agent, a simple chemical species or an enzyme, thus forming a more densely connected ensemble. Starting from a chemical gel prepared at a temperaure well above that marking the “melting” of the physical gel and cooling to room temperature, one should give the opportunity to local chain segments, in particular, to dangling chains, to engage in conformationally ordered structures albeit necessarily short ones. The latter could anyhow contribute positively to the network physical properties. We may therefore envisage two

Crescenzi et al.

additional limiting types of gels for gelatin (but, quite naturally, also for other biopolymers yielding aqueous physical gels, for example, gellan28), namely, physicalchemical gels and chemical-physical gels. To actually come close to the two types of limiting gels mentioned above, cross-linking by MTGase has been carried out for 8 h (always in PBS, pH 7) working either at 37 °C (the threshold of physical gelation for gelatin A) or at 50 °C for 1 h and then 7 h at 37 °C. In the latter case, the enzyme would promote a fast reticulation of conformationally disordered polypeptide chains in solution at 50 °C (the optimum working temperature for MGTase) and then continue its action, more slowly though, at the lower temperature. This should at least qualitatively apply also to the case of gels in which gelatin A3 is mixed with up to 20 wt % of HA or of HA-GK (see Materials and Methods). In these cases, however, an additional phenomenon comes into play, that is, chain aggregation inside the polymeric networks (see Results and Discussion, section 3.4) when the latter are swollen in pure water. In conclusion, to underline the main aspects presented above in sections 3 and 4, a few relevant experimental data are summarized in Table 3 for the various enzymatically gelling systems considered. These data point out that (1) the gel time is always quite shorter when protocol b is employed, (2) the G′ values follow the same trend except for the A3/ HA-GK system (see Results and Discussion), and (3) the maximum swelling values in PBS are larger for the mixed gelatin gels, the A3 gels being indeed poorly swellable materials insensitive to ionic strength. These aspects, including the facile synthesis of a class of chemical hydrogels using a microbial transglutaminase demonstrated in this work, have a bearing on the choice of the most appropriate gelling system for the application at which we aim, such as nucleus pulposus substitution, for which gel time, optimal working temperature, and swelling ability and rigidity of the final materials are all crucial factors. Acknowledgment. This work has been carried out with the financial support of the University of Rome, “La Sapienza” (Ateneo funds). The kind gifts of the enzyme sample from Ajinomoto Co., Japan, and of the HA sample from Fidia Advanced Biopolymers, SrL, Abano Terme, Italy, are also acknowledged. References and Notes (1) Rosiak, J. M.; Yoshii, F. Nucl. Instrum. Methods Phys. Res., Sect. B 1999, 151, 56-64. (2) Peppas, N. A.; Bures, P.; Leobandung, W.; Ichikawa, H. Eur. J. Pharm. Biopharm. 2000, 50, 27-46. (3) Mooney, D. J.; Lee, K. Y. Chem. ReV. 2001, 101, 1869-1879. (4) Kuijpers, A. J.; Engbers, G. H. M.; Feijen, J. Macromolecules 1999, 32, 3325-3333. (5) Meyer, K.; Palmer, J. J. Biol. Chem. 1934, 107, 629-634. (6) Laurent, T. C. The Chemistry, Biology and Medical Applications of Hyaluronan and its DeriVatiVes; Portland Press: London, 1998. (7) Crescenzi, V.; Francescangeli, A.; Renier, D.; Bellini, D. Biopolymers 2002, 64, 86-94. (8) Capitani, D.; Crescenzi, V.; De Angelis, A. A.; Segre, A. L. Macromolecules 2001, 34, 4136-4144. (9) Dal Pozzo, A.; Vanini, L.; Fagnoni, M.; Guerrini, M.; De Benedittis A.; Muzzarelli, R. A. A. Carbohydr. Polym. 2000, 42, 201-206. (10) de Jong, G. A. H.; Wijngaards, G.; Boumans, H.; Koppelman, S. J.; Hessing, M. J. Agric. Food Chem. 2001, 49, 3389-3393.

New Gelatin-Based Hydrogels (11) Fuchsbauer, H. L.; Gerber, U.; Engelmann, J.; Seeger, T.; Sinks, C.; Hecht, T. Biomaterials 1996, 17, 1481-1488. (12) Ohtsuka, T.; Sawa, A.; Kawabata, R.; Nio, N.; Motoki, M. J. Agric. Food Chem. 2000, 48, 230-6233. (13) Motoki, M.; Seguro, K. Trends Food Sci. Technol. 1998, 9, 204210. (14) Schorsch, C.; Carrie, H.; Clark, A. H.; Norton, I. T. Int. Dairy J. 2000, 10, 519-528. (15) Schorsch, C.; Carrie, H.; Norton, I. T. Int. Dairy J. 2000, 10, 529539. (16) Choi, Y. S.; Hong, S. R.; Lee, Y. M.; Song, K. W.; Parki, M. H.; Nam, Y. S. J. Biomed. Mater. Res. 1999, 48, 631-639. (17) Bulpitt, P.; Aeschlimann, D. J. Biomed. Mater. Res. 1999, 47, 152169. (18) Crescenzi, V.; Francescangeli, A.; Taglienti, A.; Segre, A. L.; Capitani D.; Mannina, L., manuscript in preparation. (19) Pouyani, T.; Kuo, J.; Harbison, G. S.; Prestwich, G. D. J. Am. Chem. Soc. 1992, 114, 5972-5976.

Biomacromolecules, Vol. 3, No. 6, 2002 1391 (20) Luo, Y.; Prestwich, G. D. Bioconjugate Chem. 2001, 12, 10851088. (21) Tomihata, K.; Ikada, Y. J. Biomed. Mater. Res. 1997, 37, 243-251. (22) Clark, A. H.; Ross-Murphy, S. B. Structural and Mechanical Properties of Biopolymer Gels. AdV. Polym. Sci. 1987, 83, 57-192. (23) Ross-Murphy, S. B. Polymer 1992, 33, 2622-2627. (24) Mohammed, Z. H.; Hember, M. W. N.; Richardson, R. K.; Morris, E. R. Carbohydr. Polym. 1998, 36, 15-26. (25) Remunan-Lopez, C.; Bodmeier, R. Int. J. Pharm. 1996, 135, 6372. (26) Tatham, A. S.; Larry, H.; Shewry, P. R.; Urry, D. W. Biochim. Biophys. Acta: Protein Struct. Mol. Enzymol. 2001, 1548, 187-193. (27) Flory, P. J. Proc. IUPAC, I. U. P. A. C., Macromol. Symp., 28th 1982, 507. (28) Dentini, M.; Desideri, P.; Crescenzi, V.; Yuguchi, Y.; Urakawa, H.; Kajiwara, K. Macromolecules 2001, 34, 1449-1453.

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