Structural and Rheological Properties of ... - ACS Publications

Biomacromolecules 2000, 1, 31-38

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Structural and Rheological Properties of Methacrylamide Modified Gelatin Hydrogels An I. Van Den Bulcke,† Bogdan Bogdanov,† Nadine De Rooze,† Etienne H. Schacht,*,† Maria Cornelissen,‡ and Hugo Berghmans§ Department of Organic Chemistry, Polymer Materials Research Group, Institute of Biomedical Technologies (IBITECH), University of Ghent, Krijgslaan 281, S4-bis, B-9000 Ghent, Belgium; Department of Anatomy, Embryology and Histology, Faculty of Medicines, University of Ghent, Godshuizenlaan 4, B-9000 Ghent, Belgium; and Laboratory for Polymer Research, Department Chemistry, University of Leuven, Celestijnenlaan 200F, B-3001 Heverlee, Belgium Received December 27, 1999; Revised Manuscript Received January 19, 2000

Dynamic shear oscillation measurements at small strain were used to characterize the viscoelastic properties and related differences in the molecular structure of hydrogels based on gelatin methacrylamide. Gelatin was derivatized with methacrylamide side groups and was subsequently cross-linked by radical polymerization via photoinitiation. The light treatment of methacrylamide gelatin solutions resulted in the production of hydrogel films with high storage modulus (G′). Mechanical spectra and thermal scanning rheology of the obtained hydrogels are described. The temperature scan of the network below and above melting point of gelatin allowed us to identify the respective contributions of chemical and physical cross-linkage to the hydrogel elastic modulus. The results indicate that the rheological properties of the gelatin-based hydrogels can be controlled by the degree of substitution, polymer concentration, initiator concentration, and UV irradiation conditions. 1. Introduction Gelatin is a proteinaceous material obtained by hydrolytic degradation of naturally occurring collagen.1,2 It derives in particular from the fundamental molecular unit of collagen, a triple helical structure, the tropocollagen rod. Gelatins are soluble in warm water (>40 °C), but on cooling thermoreversible transparent gels are normally formed.3 In fact, gel formation obtained by cooling gelatin aqueous solutions is accompanied by some characteristic changes which have been ascribed to a partial regain of the collagen triple-helix structure. Over the years much work has been devoted to the study of gel formation by biopolymers such as gelatin.4-11 The structures and mechanisms of the formation of the gel networks involved, and their mechanical properties are now well understood. Because of its unique gelling properties, gelatin is an attractive candidate as starting material for preparing hydrogels. Hydrogels are materials which, when placed in excess water, are able to swell and retain large volumes of water in its swollen three-dimensional structure without dissolution. Many materials of this type are considered to be biocompatible and a wide range of biomedical applications has been described. Among them are contact lenses,12 artificial tendons,12 matrices for tissue engineering,13 and drug delivery systems.14 Nevertheless, there is still a need to * Corresponding author. Telephone: +32-9-2644497. Fax: +32-92644990. E-mail: [email protected]. † IBITECH, University of Ghent. ‡ Faculty of Medicines, University of Ghent. § Department Chemistry, University of Leuven.

develop nontoxic biodegradable hydrogels for specific biomedical applications, e.g., wound treatment. As a biomaterial, gelatin displays several advantages:15 it is a natural polymer that has not shown antigenity, it is completely resorbable in vivo and its physicochemical properties can be suitably modulated. Furthermore, due to the large number of functional side groups, gelatin readily undergoes chemical cross-linking, which is very important for its use as a biomaterial, e.g., as a drug delivery system or wound dressing. For many applications there is a need for chemically crosslinked or so-called hardened gelatin materials. Since gelatin gels have a relatively low melting point, they are not stable at body temperature. Therefore, it is imperative to stabilize these gels by establishing chemical cross-links between the protein chains. A variety of hardening procedures are described in the literature.16,17 Chemical cross-linking typically utilizes bifunctional reagents such as glutaraldehyde18,19 and diisocyanates,20 as well as carbodiimides,21 polyepoxy compounds,22 and acyl azides.23 Glutaraldehyde is by far the most widely used agent, due to its high efficiency to stabilize collagen-based biomaterials and despite local cytotoxicity24 and calcification of long-term implants.25 In our previous studies,26 gelatin hydrogels were developed by making use of a polymeric cross-linker, dextran dialdehyde. The hydrogels were found to be biocompatible and biodegradable.27 The rheological properties28,29 and release characteristics30 were studied in detail. It was shown that chemical aging of the gelatin-dextran dialdehyde hydrogel occurred, influencing the release patterns as a function of

10.1021/bm990017d CCC: $19.00 © 2000 American Chemical Society Published on Web 02/10/2000

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storage time. Furthermore, immobilization of incorporated peptides or drugs was observed. We now report on a recent investigation of hydrogels based on methacrylamide-modified gelatin, a more attractive concept since chemically stable materials are formed. In a previous paper,31 the synthetic aspects of a two-step method for the cross-linking of gelatin have been described in detail. Gelatin is first derivatized by reaction with methacrylic anhydride. The gelatin methacrylamide can then be crosslinked in a subsequent step. The water-soluble gelatin obtained after derivatization, can be cross-linked by a number of suitable polymerization processes, such as redox, thermal, and UV treatment; γ-irradiation; or e-beam curing. In the present paper, cross-linking of methacrylamide modified gelatin is performed by UV irradiation in the presence of a water-soluble photoinitiator. The photo-cross-linking results in the formation of a chemical network of modified gelatin. Gelatin methacrylamide hydrogels are produced as a thin sheet or film, suitable for application onto a wound surface. Their melting point is high enough to retain their physical form at body temperature for a sufficiently long time. Since a two-step production method without additional cross-linker is used, no thorough washing is needed before use and cytotoxicity is expected to be significantly reduced (ongoing experiments). Literature data on the derivatization and subsequent crosslinkage of gelatin is rare. Introduction of radically polymerizable (meth)acrylates in polysaccharides was first described by Edman.32 Recently, Sherzer et al.33 reported on the effect of e-beam curing of gelatins derivatized by glycidyl methacrylate. Most rheological studies are focused on the physical crosslinking process in gelatin gels.34-36 Measurements of the oscillatory shear moduli are frequently used to monitor continuously the viscoelastic properties of cross-linking during the gelling process.9,37 In this paper, rheological measurements were used to characterize the mechanical properties of the chemically formed networks. The effect of degree of substitution, polymer concentration, initiator concentration, and UV irradiation time as well as storage conditions will be discussed. 2. Materials and Methods 2.1. Materials. Gelatin (type B) isolated from bovine skin by the alkaline process was kindly supplied by SKW Biosystems, Ghent, Belgium. It had an isoelectric point of ∼5 and a Bloom strength of 257. The viscosity of a 6.67% (w/v) solution at 60 °C was 4.88 mPa s, the pH was 5.69. This gelatin contains 0.35 mmol amino groups per gram, due to the lysine and hydroxylysine residues. Methacrylic anhydride (MAA) was obtained from Aldrich (Bornem, Belgium) and was used as received. Dialysis Membranes Spectra/Por 4 (MWCO 12000-14000) were obtained from Polylab (Antwerp, Belgium). 2,4,6-Trinitrobenzene-sulfonic acid (TNBS) analytical grade was purchased from Serva (Heidelberg, Duitsland) and sodium azide from Acros Organics (Pittsburgh, PA). The radical photoinitiator 1-[4(2-hydroxyethoxy)phenyl]-2-hydroxy-2-methyl-1-propan-1-

Van Den Bulcke et al.

one (Irgacure 2959) was a kind gift from Ciba Speciality Chemicals N. V.; Groot-Bijgaarden, Belgium. 2.2. Synthesis and Characterization of Modified Gelatin. Gelatin methacrylamide was prepared by reaction of gelatin with methacrylic anhydride. After dissolution of gelatin in phosphate buffer (pH 7.5) at 50 °C, methacrylic anhydride was added while vigorously stirring. After 1 h of reaction, the reaction mixture was diluted and dialyzed for 24 h against distilled water at 40 °C. The reaction product was then freeze-dried leading to a white solid. The degree of substitution (DS) is defined as the percentage of -amino groups that are modified and is determined by the Habeeb method.38 Gelatin methacrylamide with a range of degrees of substitution are prepared by analogous synthesis by changing the amount of methacrylic anhydride.31 2.3. Hydrogel Preparation. The hydrogels are prepared by radical cross-linking of solubilized gelatin derivatives. Cross-linking of the methacrylamide modified gelatin was performed in aqueous medium in the presence of a watersoluble photoinitiator, Irgacure 2959. Gelatin-methacrylamide (1.5 g) was dissolved in 10 mL of distilled water at 40 °C containing 6 × 10-4 g of photoinitiator (0.042% w/w to protein concentration). NaN3 was added as an antimicrobial agent. The warm mixture was then poured into a cast made of two glass plates separated by a silicon spacer 1 mm thick and cooled to room temperature for immediate exposition to LWUV light (10 mW/cm2) for a given period of time. An LWUV lamp model VL-400L (Vilber Lourmat, Marne La Valle´e, France) with 365 nm was used to cure the samples. The cast is stored at 4 °C for a selected period of time. After removal of the glass plates, a flexible 1 mm thick transparent film is obtained. 2.4. Rheological Measurements. Dynamic shear oscillation measurements at small strain are used to characterize the viscoelastic properties of cross-linked methacrylamidemodified gelatin hydrogel films. These rheological measurements at oscillatory shear deformation are carried out with a CSL2 rheometer (TA Instruments) using parallel rough plates of 40 mm diameter and plate-to-plate distance of 900 µm. Mechanical spectra are recorded in a constant strain mode, with a low deformation of 0.05 maintained over the frequency range of 0.01-10 Hz (rad/s) at 16 °C. The temperature dependence of the storage (elastic) modulus was determined by oscillatory shear deformation (dynamic rheological observations) with temperature scan ranging from 15 to 50 °C (heating rate 1.75 °C min-1) at constant frequency (1 Hz) and constant shear strain (γ ) 0.05, 1.88 mrad). 3. Results and Discussion Dynamic mechanical analysis, performed on the cured hydrogel films, provide quantitative information on the viscoelastic and rheological properties of the material by measuring the mechanical response of the samples as they are deformed under periodic strain. The elastic (also real or storage) modulus G′ and the viscous (also imaginary or loss) modulus G′′ are presented. In Figure 1, G′ and G′′ are charted as a function of radial frequency and such a plot is usually known as the “mechan-

Properties of Cross-Linked Gelatin Hydrogels

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Figure 1. Mechanical spectra of a gelatin gel and a cross-linked methacrylamide hydrogel (gelmod) measured at 17 °C. The gelatin solution (15% w/v) was stored at 4 °C for 3 weeks. Gelatin methacrylamide (DS ) 60%) hydrogel with polymer concentration 15% w/v, containing 0.05% w/v initiator, was UV-exposed for 30 min and stored at 4 °C for 2 days.

ical spectrum”. The data obtained for the gelatin and gelatinmethacrylamide systems are characterized by a storage modulus, G′(ω), which exhibits a pronounced plateau in the frequency range investigated, and by a loss modulus, G′′(ω), which is considerably smaller than the storage modulus. The mechanical spectra of the gelatin-based hydrogels are characteristic for a well-developed network. 3.1. Effect of Degree of Substitution. Methacrylamidemodified gelatin with different degrees of substitution were prepared as described above. In this paper, we have chosen to express the degree of substitution as the percentage of -amino groups that are modified (DS ) 7%, 21%, and 40%), but from the average molecular weight of gelatin (Mw 190 000) and the amino acid composition, we have calculated the number of methacrylamide side groups per gelatin macromolecule, being respectively 5, 14, and 27. The hydrogel films containing gelatin with a different number of vinyl side groups are evaluated by dynamic rheological measurements. For the purpose of comparison, and to allow the distinction between the respective contribution of the physical and chemical cross-linking to the hydrogel elastic modulus, non-cross-linked gelatin hydrogel films and methacrylamide-modified gelatin hydrogels were prepared in the absence (Figure 2a,b) and presence (Figure 3a,b) of photoinitiator and subsequent light treatment. Figure 2a represents the thermal scanning rheological observation for both the elastic and viscous modulus of a gelatin-methacrylamide solution after storage at 4 °C for 1 week. The obtained gel shows high G′ values at low temperatures, but on heating, the elastic and viscous modulus decrease and a crossover of G′′ and G′ occurs. The crossover temperature is attributed to the gel-sol transition temperature, and it indicates the transition from an elastic network formation to a solution. The effect of derivatization of gelatin on the physical network formation is shown in Figure 2b, only G′ is given for the sake of clarity. Gelatin hydrogels (DS ) 0%) with high G′ values are formed due to the structuring of helices. When the temperature rises above the gelatin melting point

Figure 2. (a) Dynamic rheological observations of the physical crosslinkage of gelatin methacrylamide with degree of substitution of 7%. (b) The effect of degree of substitution (DS) on the temperature dependence of the storage modulus (G′). Gelatin and gelatin methacrylamide hydrogels with polymer concentration 15% w/v were stored at 4 °C for 7 days.

(gel-sol transition temperature 25-30 °C) the elastic modulus drops rapidly to very low values, due to the breakdown of the gelatin physical network. Methacrylamidemodified gelatin hydrogels, prepared without the addition of a photoinitiator, shows lower G′ values, even below the gel-sol transition temperature, indicating that physical structuring is less pronounced when gelatin is modified with methacrylamide side groups. The higher the degree of modification, the lower the G′ modulus. Incorporation of vinyl side groups along the gelatin chains clearly interferes with the helix formation. The low values at temperatures above 30 °C indicate that in the absence of a photoinitiator, no chemical cross-links are formed. By contrast (Figure 3a,b), the light treatment of Irgacurecontaining methacrylamide-modified gelatin solutions leads to a hydrogel film with high storage moduli, both below and above the melting point of gelatin. This indicates that the presence of a photoinitiator system with UV exposure induces chemical cross-linking. The chemical cross-links are introduced by UV irradiation of the casts containing polymer solutions in almost random state. The samples were brought to room temperature and immediately exposed to UV light so that hardly any or no physical structuring could occur prior to chemical cross-linkage. te Nijenhuis7 has shown that gelatin solutions stored at room temperature are slow structuring systems and long induction periods are required in order to obtain helix formation. Chemical modification

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Van Den Bulcke et al. Scheme 1. Physical Gelation of Gelatin and Gelatin Methacrylamide

Figure 3. (a) Dynamic rheological observations of the physical and chemical cross-linkage of modified gelatin with degree of substitution of 40%. (b) The effect of degree of substitution (DS) on the temperature dependence of the storage modulus (G′). Gelatin methacrylamide hydrogels (15% w/v), containing 0.006% w/v initiator (Irgacure 2959), were UV-exposed for 30 min and stored at 4 °C for 2 days.

of gelatin with methacrylamide groups will even prolong this induction time. The formation of chemical bonds during UV exposure will further restrict or limit physical structuring. During a 30 min UV irradiation time, no additional physical structuring is likely to occur because a rise in temperature to 30˚C or higher is often observed, during UV irradiation. The contribution of physical network formation is therefore mainly obtained during storage of the cast at 4 °C, after UV curing. In Figure 3a, the viscous or loss modulus (G′′) clearly increases upon heating, demonstrating that the system becomes more liquidlike due to melting of the physical network. The elastic G′ value decreases only slightly because the contribution of the physical cross-links is limited. The storage modulus stays considerably higher than G′′, even above the gel-sol transition temperature, indicating that the elastic network is still present. It was concluded that the mechanical properties of the cross-linked methacrylamide gelatin films result from both chemical cross-linkage and physical structuring of methacrylamide-modified gelatin. The ranking of the curves according to gel strength in Figure 3b has changed when compared to Figure 2b. Loss moduli (G′′) are not shown in the following graphs for clarity reasons. Hydrogels comprising gelatin with a higher modification are able to form more chemical bonds; therefore, a denser network is obtained with a high elastic modulus and

network properties persisting above 30 °C (“melting point” of the gelatin gel). In contrast, the G′ decrease around the gel-sol temperature of gelatin is more enhanced in the case of low degrees of modification. The decrease of the modulus while heating the gel corresponds to melting of the physical cross-links between the gelatin chains. In all samples (DS 7%, 21%, and 40%) the contribution of the chemical crosslinkage was sufficient to obtain strong hydrogel films, even at high temperatures. The DS has an appreciable impact on the storage modulus G′ below and above 30 °C; moreover, the number of reactive vinyl side groups strongly influences the contribution of physical and chemical cross-linkage in an opposite way. A simplified representation of the impact of modification of the cross-linking phenomena is shown in Schemes 1 and 2. When gelatin undergoes a coil-helix transition on cooling, a strong physical network is formed. A high methacrylamide content seems to prevent the formation of a strong physical network, giving rise to hydrogels with poor mechanical properties. The cross-links of the network have a physical origin (e.g., hydrogen bonding) and are therefore sensitive to changes of temperature, pH, ionic content, etc. (nonpermanent cross-links). In Scheme 1, it is shown that the modification of the gelatin chains causes obstruction in the helix formation, therefore the number and size of the helices is reduced and consequently a weaker hydrogel is formed. Because of the thermoreversibility of the physical cross-links, the solution state is restored on heating above the gel-sol transition temperature. Scheme 2 represents the hydrogel formation by radical cross-linking after UV exposition of the vinyl derivatives in the presence of a photoinitiator. The degree of cross-linking or density of the hydrogels is shown to be dependent on its degree of vinyl substitution. In case of a low degree of derivatization, both physical and chemical cross-linking occur. Upon heating, the helices will dissolve, while the stable chemical bonds form an insoluble network. Because of a rise in temperature, the contribution of physically crosslinked gelatin melts and the molecular weight between crosslinks increases, resulting in a higher mobility of the gelatin chains and a lowering of the storage modulus at elevated temperatures. When the degree of vinyl substitution is increased, the probability of chemical network formation will compete with the probability of helix formation. Rheological



Figure 4. Effect of polymer concentration (10, 15, and 20% w/v) on the temperature dependence of the elastic modulus G′ of gelatin methacrylamide hydrogels (DS ) 7%), containing 0.006% w/v photoinitiator, UV-exposed for 30 min and stored at 4 °C for 2 days.

measurements confirmed that the presence of many covalent cross-links (high DS) impedes the formation of subsequent structuring on cooling and its storage modulus will not change when heated above 30 °C. 3.2. Effect of Polymer Concentration. As concentrations increase, intermolecular helix associations become more probable. A higher number of physical cross-links will shorten the average molecular weight between cross-links (Mc) and will cause a rise of the storage modulus at low temperatures. Moreover, it is shown in Figure 4 that polymer concentration also affects the storage modulus G′ above the gel-sol transition temperature. Since the concentration of methacrylamide side groups rises with increasing polymer concentration, more chemical cross-links per unit volume can be formed. The ultimate gel rigidity (both physical and chemical cross-linkage) is thus strongly related to the concentration of polymer present. Quantitatively, the effect of the degree of modification and polymer concentration on the chemical network formation can be clearly demonstrated by plotting the storage modulus G′ at elevated temperature (50 °C) as a function of degree of substitution (Figure 5). The dynamic modulus not only increases with higher methacrylamide content, but is also strongly effected by the total polymer concentration. In fact,

Figure 5. Effect on the chemical network formation (elastic modulus G′ measured at 50 °C) as a function of degree of substitution for different polymer concentrations (10, 15, and 20% w/v). The gelatin methacrylamide hydrogels, containing 0.006% w/v initiator, were UVexposed for 30 min and stored at 4 °C for 2 days.

the higher the polymer concentration (20% w/v), the more pronounced the impact of modification is on the rheological properties of the obtained gels. 3.3. Effect of Initiator Concentration and UV Irradiation Time. The temperature dependence of the storage modulus is strongly influenced by the initiator concentrations in the gel. An extensive drop of G′ is observed when less than 0.002% (w/v) initiator solution (0.21 mg of Irgacure 2959 per 10 mL polymer solution) is used. Hydrogels with 0.005% (w/v) initiator solution or more show a higher storage modulus above the gel-sol transition and are therefore more densely chemically cross-linked. The mechanical properties of gelatin-methacrylamide hydrogels increase with higher initiator concentration. Although, if concentrations higher than 0.025% w/v are applied, the hydrogels turn hard and brittle.

Scheme 2. Physical and Chemical Gelation of Gelatin Methacrylamide with Low and High Degree of Derivatization

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Van Den Bulcke et al.

Figure 6. Effect of initiator concentrations (0.001 f 0.05% w/v) on the temperature dependence of the elastic modulus G′ of gelatin methacrylamide (DS ) 60%) hydrogels (15% w/v), UV-exposed for 60 min and stored at 4 °C for 2 days.

Figure 8. Correlation of initiator concentration and UV irradiation time and its impact on the chemical network formation (elastic modulus G′ measured at 50 °C). Gelatin methacrylamide (DS ) 60%) hydrogels with polymer concentration 15% w/v stored at 4 °C for 2 h after UV irradiation of (b) 1 min, (0) 5 min, and (2) 30 min.

Figure 7. Effect of UV exposure time (10 f 120 min) on the temperature dependence of the elastic modulus G′ of gelatin methacrylamide (DS ) 40%) hydrogels (15% w/v), containing 0.006% w/v initiator. After UV irradiation the gels were stored at 4 °C for 3 days.

The initiator concentration (Figure 6) as well as the UV irradiation time (Figure 7) strongly affects the degree of chemical cross-linkage. Longer UV exposure times can improve the radical network formation. It is therefore very important to adjust both the amount of initiator and irradiation time in correlation with each other. It can be desirable to reduce the UV exposure time from 30 min to only a few minutes. But to obtain hydrogels with a high storage modulus G′ (>10.000 Pa), a significant increase of initiator content will be needed (Figure 8). On the other hand, the concentration of photoinitiator can be reduced drastically when a longer irradiation time (e.g., 30 min) is applied. A reduced concentration of initiator may be desired when considering later medical applications. 3.4. Effect of Storage Time. The sol-gel transition corresponds to a phenomenon whereby a cross-linking polymeric material undergoes a phase transition from a liquid to a solidlike state.9-11 Gelatin gels originate from physical interactions that are not single points on the chain, but

Figure 9. Effect of storage time (after UV irradiation) on the temperature dependence of the elastic modulus G′ of gelatin methacrylamide (DS ) 60%) hydrogels (15% w/v), containing 0.006% w/v initiator, UV-exposed for 10 min and stored at 4 °C for 5 min, 1 day, and 1 week.

correspond to more or less extended junction zones. A network is formed, and beyond the gel point, its rigidity continues to increase with increasing cross-linking density. The aging of the gelatin gel could be described by an increase of the helix size or by lateral aggregation leading to extended junction zones. Ross-Murphy5,9 suggested recently though that junction zone aggregation is not widespread, rather the helices “shuffle” at long times to increase the proportion of peptides in the ordered conformation. Figure 9 represents the elastic modulus G′ of chemically cross-linked methacrylamide modified gelatin hydrogels as a function of storage (aging) time. UV-cured hydrogel films based on modified gelatin with a degree of substitution of 60% were kept at 4 °C for various periods of time. The G′ values increased with storage time in the temperature range



different fields can be produced through the correct control of the different experimental parameters. Acknowledgment. This work was supported by the Flemish Institute for Science and Technology (IWT), the Fund for Scientific Research-Flanders (FWO), and the Belgian Government (IUAP-IV/11). The authors also express their gratitude to Ciba Specialty Chemicals for providing free samples of photoinitiator and to SKW Biosystems for providing well-characterized gelatin samples. References and Notes Figure 10. Effect of storage time before UV irradiation on the temperature dependence of the elastic modulus G′ of gelatin methacrylamide (DS ) 60%) hydrogels (15% w/v), containing 0.006% w/v initiator, UV-exposed for 30 min after storage at 4 °C for (×) 24 h, (2) 6 h, (0) 1 h, and (b) not stored before UV irradiation.

below 30 °C, but remained constant in the temperature range above 30 °C. This shows that the increase in storage modulus is due to an increasing physical structuring of gelatin chains. Since no chemical aging is observed, one can conclude that gelatin hydrogel films with stable properties (e.g., at body temperature) can be obtained, which can be suitable as drug delivery matrix for application on wound surfaces. Nevertheless, as shown in Figure 10, storage time before UV irradiation has a pronounced effect on the extent of crosslinkage. Gelatin-methacrylamide solutions containing watersoluble photoinitiator were stored at 4 °C for different periods of time before exposure to UV light. After UV curing at room temperature, the gels were treated equally. In contrast to Figure 9, an increase of G′ modulus was observed both below and above the gel/sol temperature of gelatin. Longer storage of the non-cross-linked gelatin methacrylamide hydrogel clearly enhances the degree of chemical crosslinkage. It is reasonable to assume that the increased chemical cross-linkage is obtained by a higher local concentration of the vinyl side groups caused by the helix formation of the gelatin chains. Because of this aggregation or clustering of the methacrylamide side groups, hydrogels with improved chemical cross-linking density can be formed. Further studies are in progress to confirm this hypothesis. 4. Conclusions Hydrogels based on methacrylamide-modified gelatin are attractive materials for biomedical applications. Their chemical cross-linkage leads to well controllable chemical networks. The relative contribution of the physical and chemical structuring and the overall strength of the final gel can be controlled by a proper control of the degree of substitution or the storage conditions. It is shown that chemical network formation competes with physical gelation. Chemical crosslinkage can be remarkably increased by enhanced lowtemperature storage prior to UV exposure. The time necessary for gel formation can be controlled by changing the photoinitiator concentrations. A wide range of gels with different mechanical properties and potential applications in

(1) Ward, A. G.; Courts, A. The Science and Technology of Gelatin; Academic Press: New York, 1977. (2) Veis, A. The Macromolecular Chemistry of Gelatin; Academic Press: 1964. (3) Djabourov, M.; Papon, P. Influence of thermal treaments on the structure and stability of gelatin gels. Polymer 1983, 24, 537-538. (4) Clark, A. H.; Ross-Murphy, S. B. Structural and Mechanical Properties of Biopolymer Gels. AdV. Polym. Sci. 1987, 83, 57-192. (5) Ross-Murphy, S. B. Structure and Rheology of Gelatin Gels. Imag. Sci. J. 1997, 45, 205-209. (6) Todd, A. Rigidity factor of gelatin gels. Nature 1961, 191, 567569. (7) te Nijenhuis, K. Investigation into the ageing process in gels of gelatin/water systems by the measurement of their dynamic moduli: Part I. Colloid Polym. Sci. 1981, 259, 522-530. (8) te Nijenhuis, K. Investigation into the ageing process in gels of gelatin/water systems by the measurement of their dynamic moduli: Part II. Colloid Polym.Sci. 1981, 259, 1017-1026. (9) Ross-Murphy, S. B. Structure and rheology of gelatin gels: recent progress. Polymer 1992, 33, 2622-2627. (10) Guenet, J.-M. ThermoreVersible Gelation of Polymers and Biopolymers; Academic Press: New York, 1992; Chapter 3. (11) te Nijenhuis, K. Thermoreversible networks: viscoelastic properties and structure of gels. AdV. Polym. Sci. 1997, 130, 160-194. (12) Peppas, N. A. Hydrogels in Medicine and Pharmacy; CRC Press: Bota Raton, FL, 1987. (13) Stanton, J. S.; Salik, V.; Bentley, G.; Dawnes, S. The growth of chondrocytes using gelfoam as a biodegradable scaffold. J. Mater. Sci. Mater. Med. 1995, 6, 739-744. (14) Roorda, W. E.; Bodde´, H. E.; De Boer, R. G.; Juningern H. E. Synthetic hydrogels as drug delivery systems. Pharm. Weekbl. Sci. Ed. 1986, 8, 165-189. (15) Panduranga Rao, K. Recent developments of collagen-based materials for medical applications and drug delivery systems. J. Biomater. Sci. Polym. Ed. 1995, 7, 623-645. (16) Burness, D. M.; Pouradier, J. The Hardening of Gelatin and Emulsions; Mees, C. E. K., James, T. H., Eds.; Macmillan: New York, 1966; Chapter 3. (17) Rault, I.; Herbage, F. D.; Abdul-Malak, N.; Huc, A. Evaluation of different chemical methods for cross-linking collagen gel, films and sponges. J. Mater. Sci. Mater. Med. 1996, 7, 215-221. (18) Jayakrishnan, A.; Jameela, S. R. Glutaraldehyde as a fixative in bioprostheses and drug delivery matrices. Biomaterials 1996, 17, 471-484. (19) Olde Damink, L. H.; Dijkstra, P. J.; Van Luyn, M. J.; van Wachem, P. B.; Nieuwenhuis, P.; Feijen, J. Glutaraldehyde as a cross-linking agent for collagen-based biomaterials. J. Mater. Sci. Mater. Med. 1995, 6, 460-472. (20) Olde Damink, L. H.; Dijkstra, P. J.; Van Luyn, M. J.; van Wachem, P. B.; Nieuwenhuis, P.; Feijen, J. Cross-linking of dermal sheep collagen using hexamethylene diisocyanate J. Mater. Sci. Mater. Med. 1995, 6, 429-434. (21) Ofner, C. M.; Bubnis, W. A. Chemical and Swelling Evaluations of amino group cross-linking in gelatin and modified gelatin matrices. Pharm. Res. 1996, 13, 1821-1827. (22) Sung, H.-W.; Hsu, H.-L.; Shih, C.-C.; Lin, D.-S. Cross-linking characteristics of biological tissues fixed with monofunctional or multifunctional epoxy compounds. Biomaterials 1996, 17, 14051410. (23) Petite, H.; Rault, I.; Huc, A.; Mesnache, P.; Herbage, D. J. Biomed. Mater. Res. 1990, 24, 179-188.

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(24) Speer, D. P.; Chvapil, M.; Eskelson, C. D.; Ulreich, J. Biological effect of residual glutaraldehyde in glutaraldehyde-tanned collagen biomaterials. J. Biomed. Mater. Res. 1980, 14, 753-764. (25) Khor, E.; Wee, A.; Loke, W. K.; Tan, B. L. Dimethyl sulfoxide as an anticalcification agent for glutaraldehyde-fixed biological tissue. J. Mater. Sci. Mater. Med. 1996, 7, 691-693. (26) Schacht, E.; Nobels, M.; Vansteenkiste, S.; Demeester, J.; Franssen, J.; Lemahieu, A. Some Aspects of the cross-linking of gelatin by dextran dialdhydes. Polym. Gels Networks 1993, 1, 213-224. (27) Draye, J.-P.; Delaey, B.; Van de Voorde, A.; Van Den Bulcke, A.; De Reu, B.; Schacht, E. In vitro and in vivo biocompatibility of dextran dialdehyde cross-linked gelatin hydrogel films. Biomaterials 1998, 19, 1677-1687. (28) Bogdanov, B.; Schacht, E.; Van Den Bulcke, A. Thermal and rheological properties of gelatin-dextran hydrogels. J. Therm. Anal. 1997, 49, 847-856. (29) Schacht, E.; Bogdanov, B.; Van Den Bulcke, A.; De Rooze, N. Hydrogels prepared by cross-linking of gelatin with dextran dialdehyde. React. Funct. Polym. 1997, 33, 109-116. (30) Draye, J.-P.; Delaey, B.; Van de Voorde, A.; Van Den Bulcke, A.; Bogdanov, B.; Schacht, E. In vitro release characteristics of bioactive molecules from dextran dialdehyde cross-linked gelatin hydrogel films. Biomaterials 1998, 19, 99-107.

Van Den Bulcke et al. (31) Van Den Bulcke, A.; De Rooze, N.; Schacht, E. Synthesis and characterisation of novel gelatin-methacrylamide hydrogels. Macromol. Rapid Commun., in press. (32) Edman, P.; Ekman, B.; Sjoholm, I. Immobilization of proteins in microspheres of biodegradable polyacryldextran. J. Pharm. Sci. 1980, 69, 838-842. (33) Scherzer, T.; Beckert, A.; Langguth, H.; Rummel, S.; Mehnert, R. Electron beam curing of methacrylated gelatin. I. Dependence of the degree of cross-linking on the irradiation dose. J. Appl. Polym. Sci. 1997, 63, 1303-1312. (34) Michon, C.; Cuvelier, G.; Launay, B. Concentration dependence of the critical viscoelastic properties of gelatin at the gel point. Rheol. Acta 1993, 32, 94-103. (35) Hsu, S.; Jamieson, A. M. Viscoelatic behaviour at the thermal solgel transition of gelatin. Polymer 1993, 34, 2602-2608. (36) Carnali, J. O. Gelation in physical associating biopolymer systems. Rheol. Acta 1992, 31, 399-412. (37) Ross-Murphy, S. B. Incipient behaviour of gelatin gels. Rheol. Acta 1991, 30, 401-411. (38) Habeeb, A. F. Determination of free amino groups in Proteins by trinitrobenzeneslfonic acid. Anal. Biochem. 1966, 14, 328-336.

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