- Email: [email protected]
Relationship between triple-helix content and mechanical properties of gelatin films
ARTICLE IN PRESS
Biomaterials 25 (2004) 5675–5680
Relationship between triple-helix content and mechanical properties of gelatin films A. Bigi*, S. Panzavolta, K. Rubini Department of Chemistry, University of Bologna, via Selmi, 2, Bologna 40126, Italy Received 29 October 2003; accepted 16 January 2004
Abstract This paper reports a study on the influence of the renaturation level of gelatin on the mechanical and swelling properties of gelatin films. Films at different renaturation level were obtained from gelatin samples with different Bloom index. It was verified that the triple-helix content, calculated from the values of the enthalpy of denaturation associated to the endothermal transition at about 41 C of gelatin, increases with the Bloom index. The d.s.c. data are further supported by the results of the X-ray diffraction investigation carried out on the same samples. The increase of triple-helix content provokes a significant reduction in the degree of swelling, and a remarkable improvement of the mechanical properties of the films. The elastic Young’s modulus, E, increases linearly with the renaturation level, from 3.6 to 12.0 MPa. Crosslinking with GTA 1% remarkably reduces the degree of swelling of all the samples, and induces a further increase of the Young’s modulus, which reaches values up to 27 MPa. r 2004 Elsevier Ltd. All rights reserved. Keywords: Gelatin; Renaturation level; Crosslinking; Mechanical properties; d.s.c.; Swelling
1. Introduction Collagens are the major structural proteins of most connective tissues as skin, bone and tendons, where they provide structural integrity to the tissues [1]. The peculiarity of the amino acid sequence accounts for the characteristic coiled coil structure of the collagen molecule, where three distinct polypeptide chains, each of which is coiled into a left-handed helix, are thrown into a right-handed superhelix stabilized through interchain hydrogen bonds and covalent crosslinks [1,2]. Thermal denaturation or physical and chemical degradation of collagen involves the breaking of the triplehelix structure into random coils to give gelatin. The present wide interest in gelatin is mainly due to its biodegradability. Food, pharmaceutical, and photographic industries are the main users of gelatin, which has several other technical applications. Its most frequent uses in the biomedical field include hard and soft capsules, microspheres, sealants for vascular prostheses, wound dressing and adsorbent pad for surgical *Corresponding author. Tel.: +39-051-2099551; fax: +30-0512099456. E-mail address: [email protected] (A. Bigi). 0142-9612/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2004.01.033
use, as well as three-dimensional tissue regeneration [3– 7]. At temperature of about 40 C, gelatin aqueous solutions are in the sol state and form physical thermoreversible gels on cooling. During gelling, the chains undergo a conformational disorder–order transition and partly regenerate the collagen triple-helix structure [8–10]. The process forms thermoreversible networks by associating helices in junction zones stabilized by hydrogen bonds. The network structure and the physical properties of the gelatin gels are mainly conditioned by the source and the conditions of extraction of the gelatin. Generally speaking, gelatin extracted at lower temperature is stiffer, and exhibit greater value of the Bloom index, which is a measure of the stiffness of the hydrogel [11]. Furthermore, the mechanical properties of drawn gelatin films have been related to the renaturation level of the protein, that is the triple-helix content, evaluated through differential scanning calorimetry [12,13]. The phenomenon of gelatin renaturation is of great interest also in view of developing materials with tunable mechanical properties for applicative purposes. Since gelatin is soluble in aqueous solution, gelatin materials for long-term biomedical applications must be submitted to crosslinking, which improves both the thermal
ARTICLE IN PRESS 5676
A. Bigi et al. / Biomaterials 25 (2004) 5675–5680
and the mechanical stability of the biopolymer. Among the chemical crosslinking agents, glutaraldehyde (GTA) is by far the most widely used, due to its high efficiency of collagenous materials stabilization [14]. We have analysed the thermal and mechanical properties of gelatin films prepared using gelatins with different Bloom index in order to investigate the relationship between the renaturation level of gelatin and the mechanical properties of the as-prepared films, and of the GTA crosslinked films.
2. Materials and methods Samples of type A gelatin from pig skin (Italgelatine S.p.A.) with different Bloom values, from 80 to 270, were used. Gelatin films were prepared from 5% aqueous gelatin solutions. Films were obtained on the bottom of Petri dishes (diameter=6 cm) after water evaporation at room temperature from 10 ml of gelatin solution. After air drying, some of the films were crosslinked with 10 ml of 1.0% (w/w) GTA solution in phosphate buffer at pH 7.4 for 24 h at room temperature. The crosslinked samples were then repeatedly washed with bidistilled water and air dried at room temperature.
temperature (TD) was determined as the peak value of the corresponding endothermic phenomena. The value of denaturation enthalpy was calculated with respect to the weight of air-dried gelatin. 2.3. X-ray diffraction High-angle X-ray diffraction patterns were recorded on a flat camera with a sample to film distance of 60 mm using Ni filtered CuK radiation. X-ray diffraction analysis of selected reflections was carried out by means of a Philips PW 1050/81 powder diffractometer equipped with a graphite monochromator in the diffracted beam. CuKa radiation was used (40 mA, 40 kV). The 2y range was from 5 to 40 at a scanning speed of 0.75 /min. 2.4. Swelling Gelatin films were weighted in air-dried conditions. They were then immersed in physiological solution for different periods of time. Wet samples were wiped with filter paper to remove excess liquid and weighted. The amount of adsorbed water was calculated as W ð%Þ ¼ 100
ðWW Wd Þ ; Wd
2.1. Mechanical tests Stress–strain curves of strip shaped (3 30 mm, thickness around 0.12 mm) films equilibrated in a mixture of water/ethanol in the ratio 2:3 for 72 h (constant relative humidity=75%) [9] were recorded using an INSTRON Testing Machine 4465, with a crosshead speed of 5 mm/ min, and the Series IX software package. The thickness of the samples was determined using a Leitz SM-LUX-POL microscope. The Young’s modulus E, the stress at break sb and the strain at break eb of the strips were measured in a static mode. 2.2. Differential scanning calorimetry Calorimetric measurements were performed using a Perkin Elmer DSC-7 equipped with a model PII intracooler. Temperature and enthalpy calibration was performed by using high purity standards (n-decane, benzene and indium). The measurements were carried out on known amounts of gelatin films (3–4 mg of dried sample), which had been stored in a mixture of water/ ethanol in the ratio 2:3 for 72 h (constant relative humidity=75%). The wet samples were wiped with filter paper to remove excess liquid and hermetically sealed in aluminium pans (to prevent any loss of liquid during measurements). Heating was carried out at 5 C min1 in the temperature range from 5 C to+120 C. Denaturation
where Ww and Wd are the weights of the wet and the airdried samples.
3. Results and discussion The d.s.c. plot of collagenous materials exhibits an endothermic peak associated to the helix–coil transition of collagen. The value of the denaturation enthalpy associated to this peak is related to the relative amount of triple helical structure in the samples, and is significantly lower for gelatin with respect to collagen [15,12]. The denaturation transition has been ascribed both to hydrogen bonds, which break endothermically, and to covalent crosslinks, which break exothermically [16]. Fig. 1 reports the d.s.c. plots recorded from films prepared with gelatins at two different Bloom values. It is evident the greater denaturation enthalpy associated with the transition of the sample at higher Bloom value. The values of denaturation temperature, TD, and denaturation enthalpy, DHD, obtained for the different samples are reported in Table 1. All the samples exhibit a denaturation temperature of about 41 C, in agreement with previous data [16], suggesting that the Bloom index does not affect significantly the thermal stability of the gelatin films. At variance, the increase of the DHD values on increasing the Bloom value of the films suggests a close relationship with the renaturation level. The
ARTICLE IN PRESS A. Bigi et al. / Biomaterials 25 (2004) 5675–5680
5677
Fig. 2. Renaturation level (X) of the gelatin films, calculated from the d.s.c. data, as a function of the Bloom index of the different samples. Fig. 1. Typical d.s.c. curves of the films obtained from (a) Gel-80, and (b) Gel-206.
Table 1 Denaturation temperature, TD and denaturation enthalpy, DHD, of gelatin films at different Bloom values Sample
TD ( C)
DHD (J/g)
Gel-80 Gel-170 Gel-206 Gel-238 Gel-270
4071 4171 4171 4171 4171
971 1472 1771 1971 2271
Each value is the mean of 10 determinations and is reported with its standard deviation.
renaturation level (X) was calculated as X ð%Þ ¼
DH 100; DHT
where DHT=44.0 J/g is the melting enthalpy of tendon collagen which was examined in the same conditions. Fig. 2 clearly shows the linear relationship between the Bloom index of the films and the calculated renaturation level, which reaches a maximum value of 50%. The presence of the triple helical structure in gelatin films can be detected also by high-angle X-ray diffraction analysis [17,18]. Collagenous materials display high-angle X-ray diffraction patterns characterized by the presence of two diffraction rings, which correspond to the periodicities of 1.1 and 0.29 nm characteristic of collagen molecular structure. The second diffraction ring is related to the distance between amino acidic residues along the helix, whereas the former and strongest one is related to the diameter of the triple helix. At variance, no discrete
diffraction effect can be appreciated in the high-angle X-ray patterns of denaturated collagen [18]. Partially renaturated collagen exhibits the 1.1 and 0.29 nm reflections, as well the d.s.c. endothermic peak [18]. Gelatin films display the high-angle reflections, indicative of collagen molecular structure (Fig. 3a). In particular, we analysed the integrated relative intensity of the 1.1 nm reflection as a measure of the relative triple-helix content of the films. Fig. 3b reports the integrated intensity of this reflection obtained through X-ray diffraction analysis of the different samples. The increase of the intensity of the 1.1 nm reflection on increasing the Bloom index confirms the close relationship between renaturation level and Bloom index of the samples. Gelatin is soluble in aqueous solution, and a few minutes of storage in physiological solution is sufficient to induce considerable swelling of the films, as it can be inferred from the data reported in Table 2. The results indicate that swelling reduces on increasing the triplehelix content of the samples, so that the dimensions of gel-270 can still be measured after 24 h of storage in physiological solution whereas gel-80 is completely dissolved after just 1 h in solution. The stress–strain curves recorded from the different samples have been used to evaluate the Young’s modulus, E, the stress at break, sb, and the deformation at break, eb of the gelatin films. The data are reported in Table 3. The values of eb increase with the increase of the Bloom index in agreement with a greater extensibility of the films containing greater amounts of triplehelix structure. Simultaneously, an even more relevant increase of sb occurs. As a consequence, the Young’s
ARTICLE IN PRESS A. Bigi et al. / Biomaterials 25 (2004) 5675–5680
5678
Fig. 3. (a) High-angle X-ray diffraction pattern of Gel-206. (b) Integrated intensity of the 1.1 nm reflection obtained from the diffraction patterns of the different samples.
Table 2 Swelling (% wt) of gelatin films as a function of storage time in physiological solution. Each value was determined in triplicate Sample
5 min
30 min
1h
5h
24 h
Gel-80 Gel-170 Gel-206 Gel-238 Gel-270
312737 300742 305735 264722 303752
695777 620712 627710 507712 584748
— 780716 798724 773712 779760
— 1560720 1506717 1497741 1491774
— — — — 1855723
Table 3 Strain at break, eb, stress at break, sb, and Young’s modulus, E, of the gelatin films as a function of the Bloom index Sample
eb (%)
sb (MPa)
E (MPa)
Gel-80 Gel-170 Gel-206 Gel-238 Gel-270
180730 227770 287744 300760 400720
1.370.3 1.970.6 3.570.8 3.570.5 5.170.8
3.670.3 5.270.6 8.071.0 9.570.9 1273.0
Each value is the mean of 10 determinations and is reported with its standard deviation.
modulus, E, calculated tangentially to the first region of the stress–strain curves increases. An approximately linear relationship was previously reported between the Bloom index and the compressive modulus of gelatin gels [10]. The results of this paper indicate that variations of Bloom index affect also the mechanical properties of the gelatin films. The films were crosslinked with 1% GTA, which was previously shown to produce an extent of crosslinking of about 98% [16]. GTA crosslinking induces a remarkable reduction of swelling. As a matter of fact, we verified that crosslinked samples exhibit a degree of swelling that is independent from the time of storage in physiological solution and
Fig. 4. Typical stress–strain curves recorded from gelatin films crosslinked with 1% glutaraldehyde: (a) Gel-80; (b) Gel-270.
from the renaturation level, and assumes an average value of about 110710%. The degree of swelling of crosslinked samples is the same whatever the Bloom values of the starting materials, suggesting that the major effect of GTA crosslinking on this parameter levels the differences observed among the uncrosslinked films. Furthermore, GTA crosslinking affects dramatically the stiffness of gelatin films, as previously reported [12,16]. Fig. 4 displays two typical stress–strain curves recorded from crosslinked samples having two different Bloom indexes. The values of stress at break of the crosslinked gelatin films do not appear significantly modified with respect to those obtained for uncrosslinked samples. On the other hand, GTA provokes a marked reduction of the extensibility of the films, because of the introduction of covalent crosslinks, so
ARTICLE IN PRESS A. Bigi et al. / Biomaterials 25 (2004) 5675–5680
5679
how hindered by its poor mechanical properties. On the other hand, gelatins with lower Bloom values, and with lower renaturation levels, could be more usefully employed in different applications, such as for the preparation of composites with stiffer materials, where the presence of gelatin is aimed to improve the handling and the fabrication, and to modulate the mechanical properties of the composite.
Acknowledgements This research was carried out with the financial support of MIUR, and the University of Bologna (Funds for Selected Research Topics). One of the authors (KR) carried out this research activity thank to a fellowship awarded by the Italgelatine S.p.A. Fig. 5. Plots of the values of the Young’s modulus of uncrosslinked (J) and crosslinked (m) gelatin films.
that eb assumes values smaller than 20%. As a consequence, the values of Young’s modulus of the crosslinked gelatin films are remarkably greater than those of uncrosslinked films. The values of Ec calculated from the stress–strain curves of crosslinked films are plotted in Fig. 5 against the renaturation level of the gelatin samples. For comparison, the values of E of the uncrosslinked samples are plotted in the same graph. Both E and Ec increase linearly with the increase of the renaturation level indicating that the triple-helix content affects the stiffness of both uncrosslinked and crosslinked films. It follows that gelatin samples with different Bloom indexes provide films with different renaturation degrees, mechanical properties, and swelling behaviour, which once crosslinked display a wide range of elastic modules.
4. Conclusions The results of this paper indicate a linear relationship between the Bloom index and the triple-helix content of gelatin films. The renaturation level greatly affects the mechanical and swelling properties of the films, which display increasing values of Young’s modulus and reduced degree of swelling as their triple-helix content increases. The improvement of the mechanical properties of the films is enhanced by crosslinking with GTA, which yields stable films with elastic modules up to 27 MPa. The availability of gelatins with relatively high Bloom values, together with the use of a crosslinking agent, could provide a successful answer to those biomedical problems, where gelatin application is some-
References [1] Ramachandran GN. Structure of collagen at the molecular level. In: Ramachandran GN, editor. Treatise on collagenchemistry of collagen, vol. 1. London: Academic Press; 1967. p. 103–83. [2] Brodsky B, Ramshaw JAM. The collagen triple-helix structure. Matrix Biol 1997;15:545. [3] Nimni ME, Cheung DT, Strates B, Kodama M, Sheikh K. Bioprosthesis derived from cross-linked and chemically modified collagenous tissues. In: Nimni ME, editor. Collagen, vol. 3. Boca Raton: CRC Press; 1988. p. 1–38. [4] Otani Y, Tabata Y, Ikada Y. Hemostatic capability of rapidly curable glues from gelatin poly (l-glutamic acid) and carbodiimide. Biomaterials 1998;19:2091–8. [5] Rose PJ, Mark HF, Bikales NM, Overberger CG, Menges G, Kroschwitz JI. Encyclopedia of polymer science and engineering, 2nd edn, vol. 7. New York: Wiley Interscience; 1987. [6] Digenis GA, Gold TB, Shah VP. Cross-linking of gelatin capsules and its relevance to their in vitro–in vivo performance. J Pharm Sci 1994;83:915–21. [7] Esposito E, Cortesi R, Nastruzzi C. Gelatin microspheres: influence of preparation parameters and thermal treatment on chemico-physical and biopharmaceutical properties. Biomaterials 1995;20:2009–20. [8] Veis A. The macromolecular chemistry of gelatin. New York, London: Academic Press; 1964. [9] Pezron I, Djabourov M, Leblond J. Conformation of gelatin chains in aqueous solutions: 1. A light and small angle neutron scattering study. Polymer 1991;32:3201–10. [10] Ross-Murphy SB. Structure and rheology of gelatin gels: recent progress. Polymer 1992;33:2622–7. [11] Usta M, Piech DL, MacCrone RK, Hillig WB. Behaviour and properties of net and filled gelatins. Biomaterials 2003;24: 165–72. [12] Bigi A, Bracci B, Cojazzi G, Panzavolta S, Roveri N. Drawn gelatin films with improved mechanical properties. Biomaterials 1998;19:2335–40. [13] Bigi A, Borghi M, Cojazzi G, Fichera AM, Panzavolta S, Roveri N. Structural and mechanical properties of crosslinked drawn gelatin films. J Thermal Anal 2000;61:451–9. [14] Khor E. Methods for the treatment of collagenous tissues for bioprostheses. Biomaterials 1997;18:95–103.
ARTICLE IN PRESS 5680
A. Bigi et al. / Biomaterials 25 (2004) 5675–5680
[15] Achet D, He XW. Determination of the renaturation level in gelatin films. Polymer 1995;36:787–91. [16] Bigi A, Cojazzi G, Panzavolta S, Roveri N, Rubini K. Mechanical and thermal properties of gelatin films at different degrees of glutaraldehyde crosslinking. Biomaterials 2001;22: 763–8.
[17] Tanioka A, Miyasaka K, Ishikawa K. Reconstitution of collagen fold structure with stretching gelatin film. Biopolymers 1976;15:1505–11. [18] Bigi A, Cojazzi G, Roveri N, Koch MHJ. Differential scanning calorimetry and X-ray diffraction study of tendon collagen thermal denaturation. Int J Biol Macromol 1987;9:363–7.
Biomaterials 25 (2004) 5675–5680
Relationship between triple-helix content and mechanical properties of gelatin films A. Bigi*, S. Panzavolta, K. Rubini Department of Chemistry, University of Bologna, via Selmi, 2, Bologna 40126, Italy Received 29 October 2003; accepted 16 January 2004
Abstract This paper reports a study on the influence of the renaturation level of gelatin on the mechanical and swelling properties of gelatin films. Films at different renaturation level were obtained from gelatin samples with different Bloom index. It was verified that the triple-helix content, calculated from the values of the enthalpy of denaturation associated to the endothermal transition at about 41 C of gelatin, increases with the Bloom index. The d.s.c. data are further supported by the results of the X-ray diffraction investigation carried out on the same samples. The increase of triple-helix content provokes a significant reduction in the degree of swelling, and a remarkable improvement of the mechanical properties of the films. The elastic Young’s modulus, E, increases linearly with the renaturation level, from 3.6 to 12.0 MPa. Crosslinking with GTA 1% remarkably reduces the degree of swelling of all the samples, and induces a further increase of the Young’s modulus, which reaches values up to 27 MPa. r 2004 Elsevier Ltd. All rights reserved. Keywords: Gelatin; Renaturation level; Crosslinking; Mechanical properties; d.s.c.; Swelling
1. Introduction Collagens are the major structural proteins of most connective tissues as skin, bone and tendons, where they provide structural integrity to the tissues [1]. The peculiarity of the amino acid sequence accounts for the characteristic coiled coil structure of the collagen molecule, where three distinct polypeptide chains, each of which is coiled into a left-handed helix, are thrown into a right-handed superhelix stabilized through interchain hydrogen bonds and covalent crosslinks [1,2]. Thermal denaturation or physical and chemical degradation of collagen involves the breaking of the triplehelix structure into random coils to give gelatin. The present wide interest in gelatin is mainly due to its biodegradability. Food, pharmaceutical, and photographic industries are the main users of gelatin, which has several other technical applications. Its most frequent uses in the biomedical field include hard and soft capsules, microspheres, sealants for vascular prostheses, wound dressing and adsorbent pad for surgical *Corresponding author. Tel.: +39-051-2099551; fax: +30-0512099456. E-mail address: [email protected] (A. Bigi). 0142-9612/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2004.01.033
use, as well as three-dimensional tissue regeneration [3– 7]. At temperature of about 40 C, gelatin aqueous solutions are in the sol state and form physical thermoreversible gels on cooling. During gelling, the chains undergo a conformational disorder–order transition and partly regenerate the collagen triple-helix structure [8–10]. The process forms thermoreversible networks by associating helices in junction zones stabilized by hydrogen bonds. The network structure and the physical properties of the gelatin gels are mainly conditioned by the source and the conditions of extraction of the gelatin. Generally speaking, gelatin extracted at lower temperature is stiffer, and exhibit greater value of the Bloom index, which is a measure of the stiffness of the hydrogel [11]. Furthermore, the mechanical properties of drawn gelatin films have been related to the renaturation level of the protein, that is the triple-helix content, evaluated through differential scanning calorimetry [12,13]. The phenomenon of gelatin renaturation is of great interest also in view of developing materials with tunable mechanical properties for applicative purposes. Since gelatin is soluble in aqueous solution, gelatin materials for long-term biomedical applications must be submitted to crosslinking, which improves both the thermal
ARTICLE IN PRESS 5676
A. Bigi et al. / Biomaterials 25 (2004) 5675–5680
and the mechanical stability of the biopolymer. Among the chemical crosslinking agents, glutaraldehyde (GTA) is by far the most widely used, due to its high efficiency of collagenous materials stabilization [14]. We have analysed the thermal and mechanical properties of gelatin films prepared using gelatins with different Bloom index in order to investigate the relationship between the renaturation level of gelatin and the mechanical properties of the as-prepared films, and of the GTA crosslinked films.
2. Materials and methods Samples of type A gelatin from pig skin (Italgelatine S.p.A.) with different Bloom values, from 80 to 270, were used. Gelatin films were prepared from 5% aqueous gelatin solutions. Films were obtained on the bottom of Petri dishes (diameter=6 cm) after water evaporation at room temperature from 10 ml of gelatin solution. After air drying, some of the films were crosslinked with 10 ml of 1.0% (w/w) GTA solution in phosphate buffer at pH 7.4 for 24 h at room temperature. The crosslinked samples were then repeatedly washed with bidistilled water and air dried at room temperature.
temperature (TD) was determined as the peak value of the corresponding endothermic phenomena. The value of denaturation enthalpy was calculated with respect to the weight of air-dried gelatin. 2.3. X-ray diffraction High-angle X-ray diffraction patterns were recorded on a flat camera with a sample to film distance of 60 mm using Ni filtered CuK radiation. X-ray diffraction analysis of selected reflections was carried out by means of a Philips PW 1050/81 powder diffractometer equipped with a graphite monochromator in the diffracted beam. CuKa radiation was used (40 mA, 40 kV). The 2y range was from 5 to 40 at a scanning speed of 0.75 /min. 2.4. Swelling Gelatin films were weighted in air-dried conditions. They were then immersed in physiological solution for different periods of time. Wet samples were wiped with filter paper to remove excess liquid and weighted. The amount of adsorbed water was calculated as W ð%Þ ¼ 100
ðWW Wd Þ ; Wd
2.1. Mechanical tests Stress–strain curves of strip shaped (3 30 mm, thickness around 0.12 mm) films equilibrated in a mixture of water/ethanol in the ratio 2:3 for 72 h (constant relative humidity=75%) [9] were recorded using an INSTRON Testing Machine 4465, with a crosshead speed of 5 mm/ min, and the Series IX software package. The thickness of the samples was determined using a Leitz SM-LUX-POL microscope. The Young’s modulus E, the stress at break sb and the strain at break eb of the strips were measured in a static mode. 2.2. Differential scanning calorimetry Calorimetric measurements were performed using a Perkin Elmer DSC-7 equipped with a model PII intracooler. Temperature and enthalpy calibration was performed by using high purity standards (n-decane, benzene and indium). The measurements were carried out on known amounts of gelatin films (3–4 mg of dried sample), which had been stored in a mixture of water/ ethanol in the ratio 2:3 for 72 h (constant relative humidity=75%). The wet samples were wiped with filter paper to remove excess liquid and hermetically sealed in aluminium pans (to prevent any loss of liquid during measurements). Heating was carried out at 5 C min1 in the temperature range from 5 C to+120 C. Denaturation
where Ww and Wd are the weights of the wet and the airdried samples.
3. Results and discussion The d.s.c. plot of collagenous materials exhibits an endothermic peak associated to the helix–coil transition of collagen. The value of the denaturation enthalpy associated to this peak is related to the relative amount of triple helical structure in the samples, and is significantly lower for gelatin with respect to collagen [15,12]. The denaturation transition has been ascribed both to hydrogen bonds, which break endothermically, and to covalent crosslinks, which break exothermically [16]. Fig. 1 reports the d.s.c. plots recorded from films prepared with gelatins at two different Bloom values. It is evident the greater denaturation enthalpy associated with the transition of the sample at higher Bloom value. The values of denaturation temperature, TD, and denaturation enthalpy, DHD, obtained for the different samples are reported in Table 1. All the samples exhibit a denaturation temperature of about 41 C, in agreement with previous data [16], suggesting that the Bloom index does not affect significantly the thermal stability of the gelatin films. At variance, the increase of the DHD values on increasing the Bloom value of the films suggests a close relationship with the renaturation level. The
ARTICLE IN PRESS A. Bigi et al. / Biomaterials 25 (2004) 5675–5680
5677
Fig. 2. Renaturation level (X) of the gelatin films, calculated from the d.s.c. data, as a function of the Bloom index of the different samples. Fig. 1. Typical d.s.c. curves of the films obtained from (a) Gel-80, and (b) Gel-206.
Table 1 Denaturation temperature, TD and denaturation enthalpy, DHD, of gelatin films at different Bloom values Sample
TD ( C)
DHD (J/g)
Gel-80 Gel-170 Gel-206 Gel-238 Gel-270
4071 4171 4171 4171 4171
971 1472 1771 1971 2271
Each value is the mean of 10 determinations and is reported with its standard deviation.
renaturation level (X) was calculated as X ð%Þ ¼
DH 100; DHT
where DHT=44.0 J/g is the melting enthalpy of tendon collagen which was examined in the same conditions. Fig. 2 clearly shows the linear relationship between the Bloom index of the films and the calculated renaturation level, which reaches a maximum value of 50%. The presence of the triple helical structure in gelatin films can be detected also by high-angle X-ray diffraction analysis [17,18]. Collagenous materials display high-angle X-ray diffraction patterns characterized by the presence of two diffraction rings, which correspond to the periodicities of 1.1 and 0.29 nm characteristic of collagen molecular structure. The second diffraction ring is related to the distance between amino acidic residues along the helix, whereas the former and strongest one is related to the diameter of the triple helix. At variance, no discrete
diffraction effect can be appreciated in the high-angle X-ray patterns of denaturated collagen [18]. Partially renaturated collagen exhibits the 1.1 and 0.29 nm reflections, as well the d.s.c. endothermic peak [18]. Gelatin films display the high-angle reflections, indicative of collagen molecular structure (Fig. 3a). In particular, we analysed the integrated relative intensity of the 1.1 nm reflection as a measure of the relative triple-helix content of the films. Fig. 3b reports the integrated intensity of this reflection obtained through X-ray diffraction analysis of the different samples. The increase of the intensity of the 1.1 nm reflection on increasing the Bloom index confirms the close relationship between renaturation level and Bloom index of the samples. Gelatin is soluble in aqueous solution, and a few minutes of storage in physiological solution is sufficient to induce considerable swelling of the films, as it can be inferred from the data reported in Table 2. The results indicate that swelling reduces on increasing the triplehelix content of the samples, so that the dimensions of gel-270 can still be measured after 24 h of storage in physiological solution whereas gel-80 is completely dissolved after just 1 h in solution. The stress–strain curves recorded from the different samples have been used to evaluate the Young’s modulus, E, the stress at break, sb, and the deformation at break, eb of the gelatin films. The data are reported in Table 3. The values of eb increase with the increase of the Bloom index in agreement with a greater extensibility of the films containing greater amounts of triplehelix structure. Simultaneously, an even more relevant increase of sb occurs. As a consequence, the Young’s
ARTICLE IN PRESS A. Bigi et al. / Biomaterials 25 (2004) 5675–5680
5678
Fig. 3. (a) High-angle X-ray diffraction pattern of Gel-206. (b) Integrated intensity of the 1.1 nm reflection obtained from the diffraction patterns of the different samples.
Table 2 Swelling (% wt) of gelatin films as a function of storage time in physiological solution. Each value was determined in triplicate Sample
5 min
30 min
1h
5h
24 h
Gel-80 Gel-170 Gel-206 Gel-238 Gel-270
312737 300742 305735 264722 303752
695777 620712 627710 507712 584748
— 780716 798724 773712 779760
— 1560720 1506717 1497741 1491774
— — — — 1855723
Table 3 Strain at break, eb, stress at break, sb, and Young’s modulus, E, of the gelatin films as a function of the Bloom index Sample
eb (%)
sb (MPa)
E (MPa)
Gel-80 Gel-170 Gel-206 Gel-238 Gel-270
180730 227770 287744 300760 400720
1.370.3 1.970.6 3.570.8 3.570.5 5.170.8
3.670.3 5.270.6 8.071.0 9.570.9 1273.0
Each value is the mean of 10 determinations and is reported with its standard deviation.
modulus, E, calculated tangentially to the first region of the stress–strain curves increases. An approximately linear relationship was previously reported between the Bloom index and the compressive modulus of gelatin gels [10]. The results of this paper indicate that variations of Bloom index affect also the mechanical properties of the gelatin films. The films were crosslinked with 1% GTA, which was previously shown to produce an extent of crosslinking of about 98% [16]. GTA crosslinking induces a remarkable reduction of swelling. As a matter of fact, we verified that crosslinked samples exhibit a degree of swelling that is independent from the time of storage in physiological solution and
Fig. 4. Typical stress–strain curves recorded from gelatin films crosslinked with 1% glutaraldehyde: (a) Gel-80; (b) Gel-270.
from the renaturation level, and assumes an average value of about 110710%. The degree of swelling of crosslinked samples is the same whatever the Bloom values of the starting materials, suggesting that the major effect of GTA crosslinking on this parameter levels the differences observed among the uncrosslinked films. Furthermore, GTA crosslinking affects dramatically the stiffness of gelatin films, as previously reported [12,16]. Fig. 4 displays two typical stress–strain curves recorded from crosslinked samples having two different Bloom indexes. The values of stress at break of the crosslinked gelatin films do not appear significantly modified with respect to those obtained for uncrosslinked samples. On the other hand, GTA provokes a marked reduction of the extensibility of the films, because of the introduction of covalent crosslinks, so
ARTICLE IN PRESS A. Bigi et al. / Biomaterials 25 (2004) 5675–5680
5679
how hindered by its poor mechanical properties. On the other hand, gelatins with lower Bloom values, and with lower renaturation levels, could be more usefully employed in different applications, such as for the preparation of composites with stiffer materials, where the presence of gelatin is aimed to improve the handling and the fabrication, and to modulate the mechanical properties of the composite.
Acknowledgements This research was carried out with the financial support of MIUR, and the University of Bologna (Funds for Selected Research Topics). One of the authors (KR) carried out this research activity thank to a fellowship awarded by the Italgelatine S.p.A. Fig. 5. Plots of the values of the Young’s modulus of uncrosslinked (J) and crosslinked (m) gelatin films.
that eb assumes values smaller than 20%. As a consequence, the values of Young’s modulus of the crosslinked gelatin films are remarkably greater than those of uncrosslinked films. The values of Ec calculated from the stress–strain curves of crosslinked films are plotted in Fig. 5 against the renaturation level of the gelatin samples. For comparison, the values of E of the uncrosslinked samples are plotted in the same graph. Both E and Ec increase linearly with the increase of the renaturation level indicating that the triple-helix content affects the stiffness of both uncrosslinked and crosslinked films. It follows that gelatin samples with different Bloom indexes provide films with different renaturation degrees, mechanical properties, and swelling behaviour, which once crosslinked display a wide range of elastic modules.
4. Conclusions The results of this paper indicate a linear relationship between the Bloom index and the triple-helix content of gelatin films. The renaturation level greatly affects the mechanical and swelling properties of the films, which display increasing values of Young’s modulus and reduced degree of swelling as their triple-helix content increases. The improvement of the mechanical properties of the films is enhanced by crosslinking with GTA, which yields stable films with elastic modules up to 27 MPa. The availability of gelatins with relatively high Bloom values, together with the use of a crosslinking agent, could provide a successful answer to those biomedical problems, where gelatin application is some-
References [1] Ramachandran GN. Structure of collagen at the molecular level. In: Ramachandran GN, editor. Treatise on collagenchemistry of collagen, vol. 1. London: Academic Press; 1967. p. 103–83. [2] Brodsky B, Ramshaw JAM. The collagen triple-helix structure. Matrix Biol 1997;15:545. [3] Nimni ME, Cheung DT, Strates B, Kodama M, Sheikh K. Bioprosthesis derived from cross-linked and chemically modified collagenous tissues. In: Nimni ME, editor. Collagen, vol. 3. Boca Raton: CRC Press; 1988. p. 1–38. [4] Otani Y, Tabata Y, Ikada Y. Hemostatic capability of rapidly curable glues from gelatin poly (l-glutamic acid) and carbodiimide. Biomaterials 1998;19:2091–8. [5] Rose PJ, Mark HF, Bikales NM, Overberger CG, Menges G, Kroschwitz JI. Encyclopedia of polymer science and engineering, 2nd edn, vol. 7. New York: Wiley Interscience; 1987. [6] Digenis GA, Gold TB, Shah VP. Cross-linking of gelatin capsules and its relevance to their in vitro–in vivo performance. J Pharm Sci 1994;83:915–21. [7] Esposito E, Cortesi R, Nastruzzi C. Gelatin microspheres: influence of preparation parameters and thermal treatment on chemico-physical and biopharmaceutical properties. Biomaterials 1995;20:2009–20. [8] Veis A. The macromolecular chemistry of gelatin. New York, London: Academic Press; 1964. [9] Pezron I, Djabourov M, Leblond J. Conformation of gelatin chains in aqueous solutions: 1. A light and small angle neutron scattering study. Polymer 1991;32:3201–10. [10] Ross-Murphy SB. Structure and rheology of gelatin gels: recent progress. Polymer 1992;33:2622–7. [11] Usta M, Piech DL, MacCrone RK, Hillig WB. Behaviour and properties of net and filled gelatins. Biomaterials 2003;24: 165–72. [12] Bigi A, Bracci B, Cojazzi G, Panzavolta S, Roveri N. Drawn gelatin films with improved mechanical properties. Biomaterials 1998;19:2335–40. [13] Bigi A, Borghi M, Cojazzi G, Fichera AM, Panzavolta S, Roveri N. Structural and mechanical properties of crosslinked drawn gelatin films. J Thermal Anal 2000;61:451–9. [14] Khor E. Methods for the treatment of collagenous tissues for bioprostheses. Biomaterials 1997;18:95–103.
ARTICLE IN PRESS 5680
A. Bigi et al. / Biomaterials 25 (2004) 5675–5680
[15] Achet D, He XW. Determination of the renaturation level in gelatin films. Polymer 1995;36:787–91. [16] Bigi A, Cojazzi G, Panzavolta S, Roveri N, Rubini K. Mechanical and thermal properties of gelatin films at different degrees of glutaraldehyde crosslinking. Biomaterials 2001;22: 763–8.
[17] Tanioka A, Miyasaka K, Ishikawa K. Reconstitution of collagen fold structure with stretching gelatin film. Biopolymers 1976;15:1505–11. [18] Bigi A, Cojazzi G, Roveri N, Koch MHJ. Differential scanning calorimetry and X-ray diffraction study of tendon collagen thermal denaturation. Int J Biol Macromol 1987;9:363–7.