New Hydrogels Based on Substituted Anhydride Modified Collagen

Article pubs.acs.org/IECR

New Hydrogels Based on Substituted Anhydride Modified Collagen and 2‑Hydroxyethyl Methacrylate. Synthesis and Characterization Daniela Pamfil,† Christoph Schick,‡ and Cornelia Vasile*,† †

Department of Physical Chemistry of Polymers, “Petru Poni” Institute of Macromolecular Chemistry, Romanian Academy, , 41 A, Grigore Ghica Vodă Alley, 700487, Iaşi, Romania ‡ Institute of Physics, University of Rostock, Wismarsche Strasse 43-45, 18051, Rostock, Germany S Supporting Information *

ABSTRACT: Semi-interpenetrated networks were obtained by free radical polymerization/cross-linking of substituted anhydride modified collagen with 2-hydroxyethyl methacrylate (HEMA) in the presence of ammonium persulfate (APS) and N,N,N′,N′-tetramethyl ethylene diamine (TEMED). Substituted anhydride modified collagens, porous materials with vinyl groups, have been previously synthesized by reaction of soluble collagen with dimethyl maleic anhydride (DMA) or citraconic anhydride (CTA). The structure and physicochemical properties of the obtained hydrogels were investigated by Fourier transform infrared spectroscopy, scanning electron microscopy (SEM), and water retention studies. Thermal properties of substituted anhydride modified collagen and their corresponding hydrogels have been investigated by differential scanning calorimetry (DSC) in dried and hydrated states. The denaturation temperature (Td), denaturation enthalpy, and glass transition temperature (Tg) have been determined, and they were found to be dependent on chemical composition, thermal history, and moisture content. The water states (free or bonded) in the hydrated samples were correlated with their swelling degree.

1. INTRODUCTION The use of collagen, a major component of connective tissue, has acquired an impressive prestige in many fields in past years due to its unique properties such as low immunogenicity and toxicity, excellent biocompatibility and safety owing to its biological characteristics, such as biodegradability and weak antigenecity, ability of skin, bone, or other tissue regeneration, etc. Also, collagen may gain special and improved properties by cross-linking, blending with other polymers, chemical modification, filling with solid inclusions, etc.1 Literature reports many cases of chemical modification with different low molecular weight reagents (e.g., imides, anhydrides, chloroanhydrides, acid halide, sulfonyl halide, active ester amine modifying agents, epoxy succinic acid, isocyanate)2,3 to collagen materials. Chemical hydrogels,4 three-dimensional covalently crosslinked networks of hydrophilic polymers capable of imbibing and retaining large quantities of water in their swollen structures without dissolution,5 have gained great interest in biomedical applications, such as wound care products, dental and ophthalmic materials, drug delivery systems, elements of implants, and constituents of hybrid-type organs, as well as stimuli-sensitive systems.6 Natural collagen hydrogels have structural similarity with the physiological extracellular matrix, promote cell migration, proliferation, and adhesion,7 and offer biosafe profiles, no toxicity, and no chronic inflammatory response if used for implantation as was reported in previous studies of collagen hydrogel.8 Clinically, collagen gels have been applied for the reconstruction of whole organs such as skin, blood vessels, and small intestine.9 One of the most extensively studied hydrogels in biomedical applications is poly(2-hydroxyethyl methacrylate) (pHEMA), a thermoset (does not soften when heated owing to its rigid © 2014 American Chemical Society

three-dimensional network) that exhibits a high degree of chemical and hydrolytic stability10 proving resistance to enzymatically degradation and hydrolysis by acidic or alkaline solutions.11 It also presents a tunable chemical composition; i.e., it can adopt different shapes and forms,12 has excellent biocompatibility and physicochemical properties similar to those of living tissues,5,13 and has good tolerance for entrapped cells. HEMA is a synthetic hydrophilic low-molecular-weight monomer well-known for its low toxicity14 and polar properties15 in spite of its contact allergic reactions.16 Synthetic hydrogels containing pHEMA are attractive biomaterials due to their tissue-like elasticity, high diffusion capability, and high water content.17,18 Differential scanning calorimetry (DSC) was used to study the thermal behavior of materials as they undergo physical and chemical changes during heating.19 By this technique changes in thermodynamic functions (entropy, enthalpy, and free energy) associated with such physical processes such as the glass transition, relaxation phenomena, crystallization, and melting or chemical processes such as cross-linking, degradation, etc. are evaluated. The state of the water is one of the main characteristics which define the vast applications of hydrogels in the life science arena.20 The most dominant feature of the hydrogels is the importance of water included within the network to form interactions with constituents of the hydrogels. Hence, the state of water provides useful information on the behavior of hydrogels to investigate the water−polymer interaction.21 Received: Revised: Accepted: Published: 11239

April 24, 2014 June 17, 2014 June 17, 2014 June 30, 2014 dx.doi.org/10.1021/ie5016848 | Ind. Eng. Chem. Res. 2014, 53, 11239−11248

Industrial & Engineering Chemistry Research

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mixture was obtained which was cast into polyethylene molds and left to react at room temperature for 24 h. The obtained hydrogels by cross-linking/polymerization of HEMA with DMA-modified collagen (H DMA 7.2 and H DMA 8.2) or CTA-modified collagen (H CTA 5.6 and H CTA 9.6) were purified by dialysis against deionized water for 3 days to remove the unreacted compounds. Then, these hydrogels were dried by lyophilization for 1 day and maintained in a desiccator in the presence of P2O5 for characterization. 2.3. Hydrogel Characterization. 2.3.1. FT-IR (Fourier Transform Infrared) Spectroscopy. FT-IR spectra were recorded in KBr pellets using a Bruker Vertex 70 spectrophotometer in the range 600−4000 cm−1 (resolution 2 cm −1, 32 scans), at ambient temperature. To prepare the pellets, 500 mg of KBr was carefully mixed with 5−6 mg of the sample. The processing of spectra was achieved using OPUS software version 5. 2.3.2. Scanning Electron Microscopy (SEM). The analyses were carried out using a QUANTA 200 scanning electronic microscope with integrated EDS system, GENESIS XM 2i EDAX with SUTW detector; the magnification is indicated on the figures. 2.3.3. Swelling Degree (SD) of Collagen-Based Hydrogels. The swelling behavior of hydrogels was gravimetrically determined. The swelling degree was evaluated using the following expression:

Water exists in at least two states, classified as unbound and bound water states, in the polymer network. Free water, however, does not contribute in the hydrogen bonds on the hydrogel structure, while bound water interacts more or less tightly with the hydrogel molecules. The determination of the amounts of the different types of water present in hydrogels is important for understanding the nature of adsorption/ desorption processes, for characterization of their biocompatibility, and for the engineering of gels with appropriate structural characteristics for their intended biological applications including tissue engineering.20 In our previous paper, collagen was chemical modified with citraconic anhydride (CTA) and dimethyl maleic anhydride (DMA).22 This paper deals with the obtaining and characterization of hydrogels synthesized by a free radical polymerization of these substituted anhydride modified collagens with watersoluble monomer (i.e., HEMA), in the presence of a crosslinker. A second purpose of using DSC is the determination of the water state trapped in hydrogel structures containing anhydride-modified collagen. A detailed DSC study of the substituted anhydride modified collagen and their hydrogels with HEMA in hydrated and dry states to assess both the water state in both kinds of collagen-based materials and also transitions occurring by heating is presented.

2. MATERIALS AND METHODOLOGIES 2.1. Materials. All substances were purchased from SigmaAldrich (U.K.) except the acid-soluble collagen, type I + III from bovine skin dermis, 1.21 wt % in H2O2 solution, which was supplied by Lohmann & Rauscher GmbH (Germany). Citraconic anhydride (CTA) and 2,3-dimethyl maleic anhydride (DMA) with analytical purity, N,N,N′,N′-tetramethyl ethylene diamine (TEMED), and dimethyl sulfoxide (DMSO) were used as received. 2-Hydroxyethyl methacrylate (HEMA) was purified by passing it through an inhibitor removal column (HQ, Sigma-Aldrich, U.K.), and ammonium peroxodisulfate (APS) was purified by recrystallization from a mixture of water/ methanol (1:2, v:v). Citraconic anhydride (CTA) and 2,3-dimethyl maleic anhydride (DMA) modified collagens obtained according to a procedure described in a previous paper22 with substitution degrees of CTA 5.6 or CTA 9.6 and M̅ w of 558 and 598 kDa and DMA 7.2 or DMA 8.2 and M̅ w of 876 and 1395 kDa, respectively, have registered good reactivity, enhanced solubility in acidic media and thermal stability in solid state, and increased viscosity and molecular weight in solution compared to unmodified collagen. 2.2. Procedure for Hydrogel Preparation. Copolymerization/Cross-Linking of Anhydride Modified Collagen with HEMA. Hydrogels were prepared by radical cross-linking polymerization of anhydride-modified collagens CTA 5.6 or CTA 9.6 and DMA 7.2 or DMA 8.2 in the presence of HEMA. First, HEMA was purified by passing through an ion-exchangeresin column for removing hydroquinone inhibitors and added into an aqueous solution of modified collagen under continuous stirring for homogenization. Hydrogels were synthesized by precipitation polymerization at room temperature using the redox initiator system ammonium persulfate (APS)/N,N,N′,N′-tetramethyl ethylene diamine (TEMED).23,24 A purified APS solution with a concentration of 5 wt % in deionized water was used. The hydrogels were produced with a 30 wt % modified collagen reported to HEMA and a 2 wt % APS reported to TEMED. A homogeneous

%SD = [(mt − m0)/m0]·100

(1)

where mt is the mass of the swollen hydrogel at time t and m0 is the mass of dry hydrogel at time 0. The experiment was carried out in a buffer solution (pH 7.4) at a temperature of 37 °C in order to simulate the physiological human body conditions. The kinetics of solvent diffusion into the matrixes was established by applying the equation:25

Wt /Weq = kt n

(2)

where Wt and Weq represent the amounts of solvent absorbed by the matrixes at time t and at equilibrium, respectively; k is the swelling rate constant or specific rate characteristic of the system and n is the power diffusion law exponent which takes into account the type of solvent transport. Equation 2 applies to initial states of swelling (swelling degree less than 60%) where linearity of ln Ft as a function of ln t is obtained. 2.4. Differential Scanning Calorimetry Analysis. The thermal stability of the triple helical domains of collagen, an ordered biopolymer, is directly related to the denaturation parameterstemperature and enthalpy of denaturationthat can be compared in the different structures. Differential scanning calorimetry (DSC) analyses were carried out with a heat-flux Mettler Toledo instrument (Model DSC 822e, Switzerland). The DSC curves were recorded at heating and cooling rates of 5 K/min using a two-stage mechanical cooling system. Indium (mp = 156.6 °C; ΔHf = 28.45 J/g) was used as the standard reference material to calibrate the temperature and energy scales of the instrument. Samples under study of approximately 5−10 mg were accurately weighed (±0.01 mg) and encapsulated in 40 μL flat-bottomed aluminum pans. The sealed sample pans were used to prevent the water loss in the DSC measurements. Empty pans were used as references. Investigations in the hydrated state were performed between −50 and 110 °C and in the dehydrated state between 0 and 150 11240

dx.doi.org/10.1021/ie5016848 | Ind. Eng. Chem. Res. 2014, 53, 11239−11248


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Scheme 1. Schematic Representation of Modified Collagen Reaction with pHEMA Induced by (a) Redox Initiating System and (b) Homopolymerization of HEMA Monomer

or 220 °C. The weight of all samples was checked after the DSC measurements to ensure that no water loss had occurred during the heating runs. For analysis of the DSC scans the STARe and OriginLab software were used. 2.4.1. Treatment To Obtain the Dried State of the Samples. The dehydration of the samples was performed by a preliminary scan from 20 to 80 °C at 5 K/min, and annealing was maintained at the maximum temperature of the measurement for 100 min (not shown). To allow water evaporation, the DSC pan was pierced. The dehydration process consists in breaking the bonds between the water molecules and the solid substrate.26 During the drying process free water is first eliminated. Removal of water from collagen results in the formation of interchain cross-links that are the result of condensation reactions either by esterification or by amide formation. The consequences of these changes include the failure of rehydration of severely dehydrated collagen-based materials to return the materials to 100% of the pretreatment hydration level.27 Whether this lack of complete rehydration affects phenomena such as tissue ingrowth, cell adherence, and other matrix cell interactions is still debated. 2.4.2. Treatment To Obtain the Hydrated State of the Samples. A 5 μL volume of distilled water was placed in 6−8 mg of sample, previously dried, and left overnight before DSC measurements were performed to let the water be absorbed in the sample. For many applications it is important to know the enthalpy of fusion. The enthalpy change for a given phase transition (ΔH) was found by integrating over the area in which the transition is seen to occur on the DSC plot. This change was described by the following integral: ΔH =

∫T

i

Tf

Cp dt

where the limits of integration Ti and Tf are the initial and final temperatures over which the graph is integrated; Cp is the apparent specific heat at constant pressure.28 The weight fraction of free water, nw (g), was evaluated by the method proposed by Ross (1978) from the following expression:

nw =

ΔHm ΔH w

(4)

where ΔHm is the melting enthalpy of the frozen water in the sample (J) and ΔHw is the latent heat of ice melting (333.9 J/g water). By using eq 4 the bonded water content (nuw), was determined as the difference between the total water content and the free water content (nw).29 The parameters determined for collagen-based materials include the phase transition temperature (T d), which corresponds to the maximum temperature at which there was complete denaturation of the collagen sample (peak temperature); the enthalpy of denaturation (ΔHd), which defines the amount of heat required for denaturation of materials, determined as the area of the denaturation peak;30 and the glass transition temperature (Tg), which refers to the midpoint of the section in which the heat flow shows a steplike change.31 Ice melting was read either as onset or peak temperature to evidence differences between samples, with these being considered as characteristic temperatures.

3. RESULTS AND DISCUSSION 3.1. Synthesis of Hydrogels. As already mentioned, the hydrogels of modified collagens with CTA or DMA and HEMA have been prepared by cross-linking polymerization reactions in the presence of APS/TEMED as a redox initiating system. Two modified collagens have been used in each case, namely CTA

(3) 11241



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Scheme 2. Schematic Representation of the Self-Cross-Linking/Polymerization of HEMA with Modified Collagen Chains

acid.32 Therefore, the collagen displayed mainly bands at 1633, 1561, and 1400 cm−1, characteristic of the amide I, II, and III bands, respectively. Bands at 2854 and 1455 cm−1 were also observed, which represent the stretching of −CH3 and pyrollidine rings. In the spectra of hydrogels are present many characteristic bands for pHEMA, especially in the “fingerprint” region (between 750 and 2000 cm −1 ) such as at 1727 cm −1 corresponding to the −CO stretching, a small shoulder around 1649 cm−1 assigned to CC stretching, at 1360 cm−1 for H CTA and 1365 cm−1 for H DMA as deformation vibration of CH2 and CH3 groups, a shoulder at 1246 cm−1 corresponding to CO stretching, vibration of C−O−C at 1157 cm−1, and vibration at 1021 cm−1 assigned to stretching of CO(H). Also, it showed other characteristic bands at 2991 cm−1 from antisymetric vibration of CH2 and CH3, at 2950 cm−1 from symmetric vibration of CH2 and CH3, and at 2884 cm −1 from symmetric vibration of CH2. However, the shoulder at 3100 cm−1 and the peak at 816 cm−1 corresponding to CH2 coupled with skeletal stretching in the spectrum of HEMA monomer are not present in the hydrogel spectrum; also the peak from 1649 cm−1 corresponding to −CC− was changed into a small shoulder.33 These statements prove that most of the HEMA monomer molecules had polymerized either between them or with the collagenic chains. The broad adsorption in the range around 3450 cm−1 was assigned to the stretching vibration of O−H or N−H from − NH2, −OH, or −COOH groups in hydrogels.34 The band at 1455 cm−1 attributed to rings of pyrollidine from collagen was split into 1483 and 1455 cm−1 bands, which is proof that reaction between components took place. Also, the amide II band was intensified and shifted to lower wavenumbers, from 1561 to 1549 cm−1, and the band from 1198 cm−1 has disappeared in the hydrogel spectra. These changes could be assigned to the presence of pHEMA and to its crucial role into the obtained structures. No significant differences appear between the two kinds of hydrogels, although shifts in band positions exist in the following cases: H CTA 9.6, 2955 and

5.6, CTA 9.6, DMA 7.2, or DMA 8.2, obtaining H CTA and H DMA hydrogels, respectively, according to Scheme 1. The free radical polymerization is initiated on the addition of APS/TEMED. The initiating free radicals transform modified collagen or HEMA monomer into free radicals which react with other inactivated molecules and lead to a chain propagating radical (propagation step). Termination of free radical polymerization occurs by recombination or disproportionation. The reaction scheme (Scheme 1) involves the formation of the macroradical by the grafting on the modified collagen chain of HEMA and the homopolymerization of HEMA monomer also can occur forming poly(HEMA) (pHEMA). Concomitantly, the copolymerization and cross-linking of pHEMA with functionalized collagen by the development of a semiinterpenetrated polymer network takes place. Partial cross-linking occurs during both substituted anhydride preparation22 and hydrogel preparation because of active vinyl groups of modified collagens and the presence of reactive groups in HEMA. Hydrogels are formed either by physical bonds such as hydrogen bonds between the functional groups of unmodified and modified collagen and between pHEMA chains or by chemical linkages of collagen chains with pHEMA (Scheme 2). The intermolecular physical and chemical bonds of the partial interpenetrated network of obtained hydrogels ensure a high structural integrity and consistency as the water insolubility (Figure S1 in the Supporting Information) and the superabsorbent character (see below) of the novel materials prepared. 3.2. Hydrogel Characterization. 3.2.1. FT-IR Spectroscopy. In order to identify possible interactions between the two polymers (modified collagen and HEMA) involved in hydrogels synthesis, FT-IR spectra were examined with respect to that of pure collagen (Figure S2 and Table S1 in the Supporting Information). The triple helical structure of collagen arises from an unusual abundance of three amino acids: glycine, proline, and hydroxyproline. These amino acids make up the characteristic repeating motif Gly-Pro-X, where “X” can be any amino 11242



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Figure 1. Scanning electron microscopy examination of (a) pure collagen and of hydrogels: (b) H CTA 5.6, (c) H CTA 9.6, (d) H DMA 7.2, and (e) H DMA 8.2.

2920 cm−1; H DMA 8.2, 1656, 1490, 1365, and 1163 cm−1; H DMA 7.2, 1365 cm −1 (Table S1 in the Supporting Information). 3.2.2. Scanning Electron Microscopy (SEM) Examination of Collagen-Based Hydrogels. Morphological characteristics, such as porosity and texture, have been examinated by SEM micrographs which were performed on the cross sections of the freeze-dried hydrogels (Figure 1). In the SEM image of pure collagen was observed an inhomogeneous structure with

varying pore sizes. SEM examination revealed the open porosity and interconnecting pores in hydrogels. The pores are created in the hydrogel by water sublimation resulting from the freeze-drying process or by pHEMA cross-linking and stabilization of the three-dimensional network which increase the scaffold homogeneity in accordance with results found by other authors.35 The pore dimensions were in the ranges of 30−200 μm for H CTA 5.6, 100−200 μm for H CTA 9.6, 100−300 μm for H 11243



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DMA 7.2, and 70−250 μm for H DMA 8.2. The literature data confirmed that a lower freezing temperature (−40 to −60 °C) produces more homogeneous samples than a high freezing temperature.36 In the present study, the freezing before drying of the samples was performed at temperatures of approximately −4 °C, which may cause a larger pore distribution on the hydrogel structure. According to the minor variations in the modification degree of the collagen, it cannot establish an association between the hydrogel structure and the substitution degree of CTA or DMA, although it can appreciate that modification with DMA could increase the pore size because of the two substituents present in the DMA molecule. The high obtained porosity enables the hydrogels to uptake large amounts of water as will be shown below from swelling experiments. 3.2.3. Swelling Behavior of Hydrogels. The hydrogel swelling processes involves a delicate balance between swelling and elastic forces. The collagen-based hydrogels containing hydrophilic functional groups can generate strong interactions with the swelling medium which causes hydrogel expansion, but on the other hand, cross-links prevent infinite expansion of the network by generating elastic forces.37 A high swelling capacity was characteristic for studied hydrogels, a maximum swelling degree being registered after approximately 20 min, and then the plateau was reached (Figure 2).

relaxation, three classes of diffusion mechanisms are distinguished:39 (i) case I (Fickian) diffusion, in which the rate of diffusion is much less relative to the relaxation rate (n = 0.5); (ii) case II diffusion, in which diffusion is very fast compared with the relaxation processes (n = 1); and (iii) non-Fickian (anomalous) diffusion, which occurs when the diffusion and relaxation rates are comparable (0.5 < n < 1). The values of the diffusion exponent are below 0.5 because the water penetration rate is slower than the polymer chain relaxation rate. This situation, which is classified as Fickian diffusion, is called “less Fickian” behavior (Table S2 in the Supporting Information).40 3.3. Characterization in the Dry State of Unmodified and Anhydride-Modified Collagen (Types I and III) and of Hydrogels Containing These Materials. Calorimetric analyses by DSC allow measurement of the stability of the triple helical structure of collagen molecules by monitoring the changes of the phase transitions.41,42 In the first performed run (Figure 3a) a high endothermic peak was observed, assigned to a first order transition, characteristic for the collagen denaturation in the dry state which appears at 186 °C, in the case of unmodified collagen and at higher temperatures in the case of anhydride-modified collagens (189−195 °C). This effect was due to the formation of new intermolecular hydrogen

Figure 2. Swelling profiles of collagen-based hydrogels.

Higher pore diameters lower the swelling degrees of hydrogels. Higher swelling capacities were observed for H CTA 5.6 and H DMA 8.2, with maximum swelling degrees of about 240 and 205%, respectively, which are well associated with the more increased values of bonded water contents of 34.9 × 10−2 and 35.6 × 10−2 g/g compared with the other two, H CTA 9.6 and H DMA 7.2, which have maximum swelling degrees of 148 and 180% and bonded water contents of 32.9 × 10−2 and 32.6 × 10−2 g/g (see Table S4 in the Supporting Information). The same variation is found for free water content. This behavior recommends these hydrogels as a potential scaffold for tissue engineering because, as a rule, porosity allows ingrowths of cells and migration of vascular tissue.38 3.2.4. Swelling Kinetics. Kinetic parameters of swelling are given in Table S2 in the Supporting Information. It is wellknown that, based on the relative rates of diffusion and polymer

Figure 3. DSC curves of dry unmodified collagen and dry anhydridemodified collagen recorded between 20 and 220 °C: (a) first and (b) second runs. 11244



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bonds with participation of carboxyl groups of the anhydridemodified collagens. These temperature shifts may be correlated with an increase in the thermal stability.22 The literature reports Td values of 225 and 226 °C in dried conditions, for insoluble type I collagen fibers from bovine tendon,43,44 220 °C for collagen extracted from porcine aortic tissue,45 200 °C for acidsoluble calf skin collagen type I,46 and 183 °C for soluble collagen, type III from calf skin.47 The differences are explained by collagen type and its extraction method. Figure 3b reports the DSC curves corresponding to the second run of anhydride-modified collagen materials. The denaturation endotherm has disappeared from the curves indicating that all ordered structure has been lost, proving the irreversibility of collagen denaturation which is also evidenced by the absence of any signal on cooling. The denaturation phenomenon on heating, distinct from degradation, implies the rupture of interchain hydrogen bond leading to the transformation of unfolded triple helix to a random chain structure. Collagen after denaturation behaves as an amorphous polymer, called gelatin. The only phenomenon observed in all curves is a specific transition indicative of a glass transition process (Tg), lower than Td of collagen, which is associated with gelatin. The values specific for gelatin glass transition obtained in this work (165−190 °C) are close to the original value of 196 °C representative for the Tg of highly dehydrated gelatin reported in the literature.48 Enthalpies of transition in the case of hydrogels vary in a narrow range (1.47−2.26 J/g), suggesting that they require the same amount of energy to be denatured (Table S3 in the Supporting Information). Several differences in properties were observed between modified collagens and hydrogels, as detailed below. By copolymerization of modified collagen with HEMA, the denaturation temperature presented a significant decrease which can be caused by the formation of homopolymers during cross-linking (Figure 4a).49 Also, a decrease in the denaturation enthalpy and glass transition values compared to those of modified collagen materials was observed (Table S3 in the Supporting Information). In the dry state, Td,collagen < Td,modified collagen > Td,hydrogels and Tg,gelatin < Tg,modified collagen > Tg,hydrogels. There was a higher stability for modified collagen in accordance with previously presented data.28 3.4. DSC Results on Unmodified and AnhydrideModified Collagen and on Hydrogels Based on Anhydride-Modified Collagen and HEMA in Hydrated State. The reason for which similar experiments were also performed on hydrated collagen was especially to investigate the bonded/free water state in the hydrogels, in view of the strong dependence of the their properties on the moisture content and type of water.20,21 The denaturation parameters, very distinct from those obtained for dry collagen, that were found for type I + III collagen in hydrated state, were Tdh = 65 °C and ΔHdh = 58.2 J/g (Figure 5), which are close to the literature data of approximately 64 °C.50 The large endothermic peak between −20 and 10 °C corresponds to freezable water. Tdh values for collagen-based materials in the hydrated state appear at lower values compared to the materials in the dry state (Figure S3 in the Supporting Information) because of the plasticization effect of water.43,51 The DSC curves of anhydride-modified collagen (Figure S3 in the Supporting Information) look almost identical with the

Figure 4. DSC curves of dry hydrogels based on anhydride-modified collagen and HEMA recorded between 0 and 220 °C: (a) first and (a) second runs.

Figure 5. DSC thermograms of unmodified collagen recorded between −50 and 100 °C.

DSC curves of unmodified collagen, with small differences in the transition temperature (Tdh), described in Table 1. The increase of denaturation temperatures of modified collagens with respect to that of collagen was explained by their partial cross-linking through vinyl groups present in substituted anhydride molecules.22 A decrease in the denaturation temperatures by copolymerization/cross-linking with HEMA is expected because the cross-links are longer and more flexible 11245



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The hydrogel networks show a decrease in the bonded water content (from 0.5−0.54 to 0.32−0.36 g/g) and an increase in the free water content, contrary to collagen, probably due to the employment of hydrophilic groups in the formation of the polymer network knots (see Table S4 in the Supporting Information, in which are presented the values obtained for the amount of bounded and unbounded water from collagen-based materials). All thermal characteristics of the ice melting process, namely, melting enthalpy and onset and peak temperatures of the substituted anhydride modified collagen, are close to those of collagen, while a significant increase was observed for all these values corresponding to hydrogels; that should mean a strong interaction of water uptaken in the hydrogel network. This is supported by high values of the bonded water which kept approximately the same values in the second heating run, while the amount of bonded water found for other samples decreased in the second run. In a future paper the mechanical properties and degradability behavior of hydrogels will be presented.

Table 1. Transition Parameters in Hydrated State from DSC for Unmodified and Modified Collagen Materials, and for Hydrogels Containing Anhydride-Modified Collagena first run

second run

sample name

ΔHdh [J/g]

Tdh [°C]

ΔHdh [J/g]

Tdh [°C]

collagen CTA 5.6 CTA 9.6 DMA 7.2 DMA 8.2 H CTA 5.6 H CTA 9.6 H DMA 7.2 H DMA 8.2

58.2 55.21 52.17 50.07 48.72 2.12 4.43 2.9 1.75

65 64 62 62 58 41 47 51 42

15.17 19.75 19.44 16.88 18.14 − − − −

32 34 34 32 31 − − − −

ΔHdh, denaturation enthalpy in the hydrated state; Tdh, denaturation temperature in the hydrated state.

a

than those formed by vinyl bonds; therefore, the chain can more easily reorder. Some important differences can be remarked in the enthalpy of denaturation (ΔHdh) in the first run as described in the following relation: ΔHdh of pure collagen (58.2 J/g) > ΔHdh of CTA-modified collagens (55.21 and 52.17 J/g) > ΔHdh of DMA-modified collagens (50.07 and 48.72 J/g) > ΔHdh of hydrogels (1.75−4.43 J/g). The lower values in the enthalpy registered for modified collagens suggest a lower stability compared to pure collagen. At this hydrated state (60−65%) the hydrogels seem to be less stable than collagen and modified collagens. However, at a higher level of hydration both collagen and its substituted anhydride modified forms become soluble in water or acidic pH, while hydrogels remain stable (see Figure S1 in the Supporting Information). Therefore, they are stable forms which can be used for medical purposes. In the second run, this relation was maintained with the specification that the ΔHdh values had decreased and in the case of hydrogels no enthalpy was registered. The ice melting from DSC curves of hydrogels is represented by a very large peak in contrast to the peak for the denaturation process. Thus, the Td after the first scan is more decreased than the Td of simple collagen and is represented by a very small endothermic peak (including a small enthalpy), meaning that the obtained hydrogels are less thermal resistant (Table 1 and Figure S4a in the Supporting Information). On reheating, the hydrogel denaturation is irreversible as the complete disappearance of the peak corresponding to the transition temperature was observed (see Figure S4b in the Supporting Information). The ice melting is widely used to quantify the amount of freezable water in hydrated proteins. From literature data, the percent of bonded water in the case of insoluble type I collagen fiber extracted from bovine tendon was found equal to 40% with respect to total water content or 0.4 g/g.43 High values of bonded water were recorded in the first scan for modified collagens, approximately 0.5−0.54 g/g, indicating the presence of a larger amount of polar groups which gives strong hydrophilic character to modified biopolymers. Furthermore, the amount of bonded water is bigger in the second heating (0.52−0.66 g/g), meaning that after the first heating a part of free water becomes bonded. The ice fusion enthalpy values obtained in the second scan, for all the samples, are smaller than those obtained in the first scan.

4. CONCLUSIONS Modification of biological polymers is one of the most important strategies used in the fabrication of tissue engineered products. The chemical functionalization of collagen with DMA and CTA was projected in order to provide reactive sites on collagen molecules that can be copolymerized/cross-linked with HEMA. Thus, hydrogels based on pHEMA were successfully synthesized and characterized by FT-IR spectroscopy, SEM, swelling behavior, and DSC by comparing them with modified collagen products. The hydrogels show a porous structure and swell very fast. The integrity of collagen in different kinds of structures was analyzed, avoiding the extrinsic response of water by DSC. By chemical modification of collagen with anhydrides, an increase in the denaturation temperature (Td) was registered, indicating an improvement in thermal stability. This effect was caused by the formation of new intermolecular hydrogen bonds because of incorporation of carboxyl groups. As expected, the absence of water led to an increase in the Td of the dried collagen compared to the hydrated one, with water playing a plasticizing role. The hydrated cross-linked samples showed a very slight signal assigned to the denaturation enthalpy. The sample denaturation is irreversible inasmuch as the signal attributed to Td completely disappeared on reheating. It was observed that the hydrogel network formation leads to a decrease in the bonded water content and to an increase of free water content due to involvement of hydrophilic groups in the formation of the polymer network. Also, by cross-linking the water binding capacity has increased. The analysis of water states evidenced the strong hydrophilic character of the collagen materials, giving them the possibility to be used as biomaterials.

■

ASSOCIATED CONTENT

S Supporting Information *

The aspect of hydrogels is shown in Figure S1. Information regarding the characterization by FT-IR spectroscopy is detailed in Figure S2 and Table S1; also kinetic parameters of swelling for collagen hydrogels are presented in Table S2. Supplementary data concerning the investigations on differential scanning calorimetry (DSC) in dried and hydrated states are given in Tables S3 and S4 and Figures S3 and S4. This 11246



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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +40 232 217454. Fax: +40232 211299. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge the financial support by COST Action FA0904 through a STSM in the Institute of Physics, University of Rostock.

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REFERENCES

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New Hydrogels Based on Substituted Anhydride Modified Collagen

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