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Immobilisation of sulphated hyaluronan for improved biocompatibility
www.elsevier.nl/locate/jinorgbio Journal of Inorganic Biochemistry 79 (2000) 119–125
Immobilisation of sulphated hyaluronan for improved biocompatibility Rolando Barbucci *, Agnese Magnani, Roberto Rappuoli, Stefania Lamponi, Marco Consumi C.R.I.S.M.A. and Department of Chemical and Biosystem Sciences and Technology, University of Siena, Pian dei Mantellini, 44-53100 Siena, Italy Received 17 April 1999; received in revised form 17 December 1999; accepted 23 December 1999
Abstract Hyaluronan (Hyal) was modified by the insertion of sulphate to hydroxyl groups. A series of heparin-like compounds with controllable properties was obtained. The physicochemical and biological behaviours of all these sulphated hyaluronan acids (HyalSx) and their complexes with heavy metal ions (Cu2q and Zn2q) were investigated. HyalSx derivatives showed a good anticoagulant activity and low platelet aggregation which increased with increasing degree of sulphation. Moreover HyalSx and their Cu2q complexes were demonstrated to favour the growth of human endothelial cells. However, the utilisation of HyalSx as a material is hindered by its high solubility in physiological solution. Our approach to improve its stability was directed to the synthesis of new HyalSx-based hydrogels and on the preparation of new biocompatible polymeric surfaces obtained through covalent photoimmobilisation of HyalSx. The reaction of primary ovine chondrocytes and B10D2 endothelial cells was studied on both matrices in terms of cell number, F-actin and CD44 receptor immunostaining. Analysis of cell movement showed that the cells respond to HyalSx showing good adhesion and spreading. These results suggest that HyalSx containing materials could be used as biomaterials to aid cartilage repair and vessel endothelisation. q2000 Elsevier Science Inc. All rights reserved. Keywords: Sulphated hyaluronan; Hydrogels; Photoimmobilisation process; Cell mobilisation
1. Introduction Hyaluronan (Hyal) is a linear polysaccharide with a relative molecular mass in the order of millions. The molecule consists of alternating 1,4 linked units of 1,3-linked glucuronic acid and N-acetylglucosamine (Fig. 1). It is one of several glycosaminoglycan components of the extracellular matrix of connective tissue. Its remarkable viscoelastic properties account for its importance in joint lubrication and its complete lack of immunogenicity makes it an ideal building block for biomaterials needed for tissue engineering and drug delivery systems [1].
2. Sulphated hyaluronan: physicochemical and biological properties There are a number of reasons for the recent attention given to derivatise hyaluronan. Four groups on the hyaluronan molecule are available for chemical modification: carboxylate, acetamido, the reducing end group, and hydroxyl groups. The bulk of all effort to date has been directed at synthesising medically useful hyaluronan derivatives by using the * Corresponding author. Tel.: q39-0577-232-083; fax q39-0577-232033; e-mail: [email protected]
Fig. 1. Disaccharide unit of Hyal.
hydroxyl and/or the carboxylate group as the point of chemical attack. We modified Hyal by sulphation of the hydroxyl groups using the Py–SO3 complex [2]. By changing the experimental conditions and in particular the sulphating agent/polysaccharide ratio, a series of sulphated hyaluronans (HyalSx) was obtained with a degree of sulphation ranging from 1 SO3y to 4 SO3y groups for a disaccharide unit. The biological activity of HyalSx compounds has been widely evaluated, particularly regarding their heparin-like behaviour [3]. The anticoagulant activity was found to be strictly correlated to their degree of sulphation and to increase with increasing number of sulphated groups. The ability to inactivate thrombin is mainly a direct consequence of the electrostatic interaction with thrombin and does not occur via ATIII as it does for heparin. Moreover, all HyalSx compounds exhibited poor antithrombotic activity, i.e. a modest FXa inactivation was observed [3]. The presence of a large density of negative charges in these macromolecules induces a strong electrostatic repulsion with
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the negatively charged platelet membranes, therefore increasing with the number of sulphated groups. Platelet aggregation is always inhibited by HyalS4 carrying the highest number of negative charges [4].
The platelet activation is connected with specific interaction between the macromolecule and the receptors on the platelet surface. Thus, it is not dependent on the polysaccharide degree of sulphation, but the stimulation of the a-granule
Fig. 2. Structure of the polysaccharide–metal complexes.
Fig. 3. Cross-linking reaction of Hyal.
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is low for all the compounds studied, the lowest being for HyalS3 [4]. The sulphated derivatives of Hyal did not affect the growth of human umbilical vein endothelial cells. The number of cells in medium containing HyalS increased with time. A slightly better growth was observed compared to medium containing Hyal which was similar to that observed for the control. Moreover, endothelial cells in medium containing HyalS spread with no morphological alteration and without structural changes in cell organisation [5]. This behaviour accounts for the angiogenetic effect induced by these macromolecules and particularly by the Cu2q complexes with HyalSx. This is not surprising because the Cu2q heparin complex is also capable of inducing angiogenesis in vitro and in vivo [6]. Indeed, HyalS3.5 can bind several metal ions by its several coordinating groups. The HyalS3.5 macromolecule coordinates Zn2q and Cu2q ions through carboxylate and acetamido moieties without any involvement of the sulphate groups (Fig. 2). Thermodynamic and spectroscopic studies showed the type of complexes formed with Cu2q and Zn2q ions in aqueous solution [7,8]. The complex species found in aqueous solution for all the HyalS3.5–Cu(II) systems as a function of pH are the following: CuL2.5y between pH 3.0 and 6.5, and Cu(OH)2L4.5y at pH)6.0. In the case of HyalS3.5– Zn(II) only one complex species [Zn(OH)2L4.5y] is stable at pH)6.5. As HyalS is water soluble, it is clear that the application of the biological performance of HyalS requires preparations with a longer residence time or greater solidity and elasticity. Further chemical derivatisation of sulphated hyaluronan
Fig. 4. FT-IR-ATR spectra of the Hyal gels at different degrees of crosslinking.
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will provide the opportunity to develop materials for new applications.
3. Hydrogels Several different methods have been used to cross-link hyaluronan [9–12]. By controlling the extent of cross-linking, and the type of covalent bond and the hyaluronan functional group involved, it is possible to create a wide range of physically different materials. Most Hyal polymers are crosslinked via the hydroxyl groups of the hyaluronan chain, leaving the carboxylate and the acetamido groups unreacted. We adopted the strategy to utilise di-amine (such as 1,3-diaminopropan, 1,6-diaminoesan, O,O9-bis(2-aminopropyl)polyethylene glycol 500 and O,O9-bis(2-aminopropyl)polyethylene glycol 500) as cross-linking agents, once the carboxylate groups are activated by 2-chloro-1-methylpyridinium iodite (CMPJ) [13] (Fig. 3). The reaction was performed maintaining the amount of carboxylate activating agent (CMPJ) lower than the number of carboxylate groups present in the weighed quantity of Hyal. Solid-state 13C NMR [14] and FT-IR-ATR (Fig. 4) analysis showed that the quantity of carboxylate groups involved in the cross-linking reaction is regulated by the quantity of CMPJ added to the solution, with the di-amine present in large excess. This allows the physical properties of Hyal to be controlled with-
Fig. 5. Swelling degree (Sw.D.sWw/Wd=100; Wwsweight of wet sample Wdsweight of dry sample) and dynamic elastic modulus (G9) of different Hyal hydrogels.
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Fig. 6. Effect of Cu2q ions on FT-IR spectra of cross-linked Hyal.
Fig. 7. Conductivity experiment in cross-linked HyalS–Cu(II) system.
out unacceptable loss in biocompatibility. The retention of the carboxylate group is especially important because the polyanionic character of Hyal is critical to its physicochem-
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ical and biological properties. We obtain compounds ranging from highly elastoviscous solutions to viscoelastic gels and solids when the cross-linking degree varies from 5 to 100%.
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In addition, the Hyal gels can be sulphated under heterogeneous conditions to improve their haemocompatibility. The rheological analysis indicated a gel-like behaviour of these compounds, being strain independent in small deformation regions. The elastic modulus of the sulphated gels is always higher than that of the non-sulphated ones, indicating that these groups stabilise the gel structure. However, the overall mechanical properties are modulated by the type and length of cross-linking agent. The sulphation reaction did not seem to produce a large effect on the swelling and on the rheological properties (elastic modulus G9) of Hyal crosslinked with the hydrophilic PEG di-amine (Fig. 5). The sulphated gels are able to bind Cu2q ions and the coordinating groups seem similar to those found with the free HyalSx in solution as measured by the FT-IR analysis of the gel before and after the addition of Cu2q. In fact, the IR spectral analysis showed that the introduction of Cu2q induced a shift to lower wavenumber of the peaks assigned to C_O stretching at 1678 and 1660 cmy1 (due to the amide groups of both HyalSx and cross-linking bond), and to the peak at 1625 cmy1 assigned to COOy stretching (Fig. 6). This proves the involvement of all these chemical groups in the Cu2q coordination. The efficacy of these gels to bind Cu2q ions is also well documented by the decrease of conductivity when Cu2q is added to the gel. Fig. 7 shows the conductivity assay of sulphated hydrogel swollen in MOPS (3-(N-morpholino)propansulphonic acid) buffer compared with the MOPS buffer controls [15]. The arrows on the graph show the addition point of the Cu2q solution; the conductivity increase is lower in the hydrogel than in the controls, showing the coordinating ability of hyaluronan, also in cross-linked form. From a biological point of view, the sulphated gels show excellent biocompatibility and a good response to both platelets and primary chondrocytes, which would favour their utilisation in the blood-compatible materials domain and in cartilage repair. The SEM micrograph of Hyal hydrogel after contact with PRP (platelet-rich plasma) shows a very low degree of platelet adhesion. Platelets are also absent inside the mesh of the sample (Fig. 8).
Fig. 8. SEM micrograph of platelet adhesion on Hyal hydrogel. Platelets are also absent inside the meshes of the hydrogel.
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4. Photoimmobilised HyalS In order to immobilise HyalS on a polymer surface, the photoimmobilisation method developed by Matsuda and Sugawara was used [16,17]. As the method does not need the presence of functional groups on the matrix surface to connect the functional unit with the polymer, it has been applied for surface functionalisation of conventional polymers. The photoreactive polymer was obtained by a reaction of HyalS and 4-azidoaniline using water-soluble carbodiimmide (WSC) as activating agent (Fig. 9) [18]. The aqueous solution of the derivatised HyalS was cast onto the polymer substrate poly(ethylene terephthalate) (PET) and irradiated with an UV lamp. The interaction with blood of the HyalS photoimmobilised surface was evaluated in terms of thrombus formation. The amount of thrombus formed in 1 h was about 25% of that formed on the polymer substrate without immobilisation. In
Fig. 9. Preparation of azidophenylamino-derivatised HyalS (AzPhHyalS).
Fig. 10. Photoimmobilisation of AzPhHyalS on PET.
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addition, no thrombus was detected in the initial 20 min. This behaviour corresponds to that of immobilised heparin. Biological performances, in terms of adhesion and proliferation of B10D2 endothelial cells obtained from lung mouse in the HyalS immobilised PET, were compared with the corresponding non-sulphated surface. Results showed that the presence of sulphate groups on the surface is able to induce
Fig. 11. Chondrocytes immunostaining: (a) F-actin on HyalS, (b) CD44 receptor on HyalS, (c) CD44 receptor on PET.
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a better cell proliferation. With the same technique, patterned surfaces were obtained using a photomask between the UV light and the surface containing azido-HyalS (Fig. 10). Strips with different widths were obtained on changing the photomask sizes [18]. These surfaces were used to study the behaviour of B10D2 endothelial cells and primary chondrocytes and an analysis of cell movement on these substrates was performed. Most of the cells migrated from untreated strips to HyalS ones. In particular, F-actin immunostaining of chondrocytes on HyalS showed that the cells were spread and adhered on this substrate. Moreover, CD44 receptor immunostaining appeared more intense in light for cells on HyalS than for the in situ chondrocyte adhesion pathway (Fig. 11).
Fig. 12. Endothelial cells (B10D2) on photoimmobilised HyalS–PET strips 10 mm (a) and 100 mm wide (b). The preference and spreading of cells for HyalS strips was higher in 10 mm strips than in 100 mm ones, demonstrating the role of topographical cues in cell movement.
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Migration processes of B10D2 endothelial cells were studied on two different HyalS patterned surfaces, 10 and 100 mm wide. The preference and spreading of cells for HyalS strips were higher in 10 mm strips than in 100 mm ones, proving the role of topographical cues in cell movement (Fig. 12).
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ence the migration of primary chondrocytes and B10D2 endothelial cells, so that their utilisation as a scaffold for production of a tissue-engineered cartilage can be hypothesised.
Acknowledgements 5. Conclusions The hyaluronic acid was sulphated and several samples with different sulphation degrees were obtained. The sulphated groups provided the Hyal macromolecule with peculiar characteristics in terms of polyelectrolyte behaviour: the HyalS macromolecule remained in the stretched conformation at any pH value of the solution, whereas the Hyal coiled once the carboxyl groups were protonated (pH-4.0). The insertion of sulphate groups also provided the macromolecule with anticoagulant properties, resistance to enzymatic digestion, and ability to inhibit the platelet adhesion and aggregation. HyalS derivatives were able to form complexes with Cu(II) and Zn(II) ions and these complexes, in particular the Cu(II)–HyalS complex, were demonstrated to favour endothelial cell adhesion and migration. Considering the good biological properties of these modified macromolecules, new biomaterials were synthesised using HyalS as one of the components. Hydrogels with different physicochemical and mechanical properties were obtained by cross-linking the Hyal chains. The ability of the sulphated gels to complex Cu2q ions was demonstrated and the same coordinating groups as those of the free HyalS seemed to be involved in the complex formation. The sulphated gels showed an excellent biocompatibility and a good response to both platelet and primary chondrocytes, so that their utilisation in the blood-compatible materials domain in cartilage repair can be envisaged. Micropatterned surfaces with improved thromboresistance were obtained by photoimmobilisation of HyalS on PET substrates. The 10 m micropatterned surfaces were able to influ-
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We thank the ‘Progetto Finalizzato MSTA II’ of the National Research Council for financial support and the ‘Center for Cell Engineering’ (University of Glasgow, UK) for cell movement and conductivity studies.
References [1] T.C. Laurent, The Chemistry, Biology and Medical Applications of Hyaluronan and Its Derivatives, vol. 72, Portland Press, London, 1998. [2] R. Barbucci, S. Lamponi, R. Rappuoli, A. Magnani, Polym. Int. 46 (1998) 225. [3] A. Magnani, A. Albanese, S. Lamponi, R. Barbucci, Thromb. Res. 81 (1996) 383. [4] R. Barbucci, S. Lamponi, A. Magnani, L.F. Poletti, N.P. Rhodes, M. Sobel, D.F. Williams, J. Thromb. Thrombolys. 6 (1998) 109. [5] G. Abatangelo, R. Barbucci, P. Bruni, S. Lamponi, Biomaterials 18 (1997) 1411. [6] G. Alessandri, K. Raju, P.M. Gullino, Cancer Res. 43 (1983) 1790. [7] R. Barbucci, A. Magnani, M. Casolaro, N. Marchettini, C. Rossi, M. Bosco, Gazz. Chim. Ital. 125 (1995) 169. [8] A. Magnani, V. Silvestri, R. Barbucci, Macromolecular Chemistry and Physics, in press. [9] K. Tomihata, Y. Ikada, Biomaterials 18 (1997) 189. [10] K. Tomihata, Y. Ikada, J. Biomed. Mater. Res. 37 (1997) 243. [11] S. Takigami, M. Takigami, G.O. Philips, Carbohydr. Polym. 22 (1993) 153. [12] K.P. Vercruysse, D.N. Marecak, J.F. Marecek, G.D. Prestwich, Bioconjugate Chem. 8 (1997) 686. [13] T. Mukaiyama, Angew. Chem., Int. Ed. Engl. 18 (1979) 707. [14] M. Delfini, personal communication. [15] M. Mezna, A.J. Lawrence, Anal. Biochem. 218 (1994) 370. [16] T. Matsuda, T. Sugawara, Langmuir 11 (1995) 2272. [17] N. Patel, R. Padera, G. Sanders, S. Cannizzaro, M. Davies, R. Langer, C. Roberts, S. Tendler, P. Williams, K. Shakesheff, J. FASEB 12 (1998) 1447. [18] G. Chen, Y. Ito, Y. Imanishi, A. Magnani, S. Lamponi, R. Barbucci, Bioconjugate Chem. 8 (1997) 730.
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Article: 6349
Immobilisation of sulphated hyaluronan for improved biocompatibility Rolando Barbucci *, Agnese Magnani, Roberto Rappuoli, Stefania Lamponi, Marco Consumi C.R.I.S.M.A. and Department of Chemical and Biosystem Sciences and Technology, University of Siena, Pian dei Mantellini, 44-53100 Siena, Italy Received 17 April 1999; received in revised form 17 December 1999; accepted 23 December 1999
Abstract Hyaluronan (Hyal) was modified by the insertion of sulphate to hydroxyl groups. A series of heparin-like compounds with controllable properties was obtained. The physicochemical and biological behaviours of all these sulphated hyaluronan acids (HyalSx) and their complexes with heavy metal ions (Cu2q and Zn2q) were investigated. HyalSx derivatives showed a good anticoagulant activity and low platelet aggregation which increased with increasing degree of sulphation. Moreover HyalSx and their Cu2q complexes were demonstrated to favour the growth of human endothelial cells. However, the utilisation of HyalSx as a material is hindered by its high solubility in physiological solution. Our approach to improve its stability was directed to the synthesis of new HyalSx-based hydrogels and on the preparation of new biocompatible polymeric surfaces obtained through covalent photoimmobilisation of HyalSx. The reaction of primary ovine chondrocytes and B10D2 endothelial cells was studied on both matrices in terms of cell number, F-actin and CD44 receptor immunostaining. Analysis of cell movement showed that the cells respond to HyalSx showing good adhesion and spreading. These results suggest that HyalSx containing materials could be used as biomaterials to aid cartilage repair and vessel endothelisation. q2000 Elsevier Science Inc. All rights reserved. Keywords: Sulphated hyaluronan; Hydrogels; Photoimmobilisation process; Cell mobilisation
1. Introduction Hyaluronan (Hyal) is a linear polysaccharide with a relative molecular mass in the order of millions. The molecule consists of alternating 1,4 linked units of 1,3-linked glucuronic acid and N-acetylglucosamine (Fig. 1). It is one of several glycosaminoglycan components of the extracellular matrix of connective tissue. Its remarkable viscoelastic properties account for its importance in joint lubrication and its complete lack of immunogenicity makes it an ideal building block for biomaterials needed for tissue engineering and drug delivery systems [1].
2. Sulphated hyaluronan: physicochemical and biological properties There are a number of reasons for the recent attention given to derivatise hyaluronan. Four groups on the hyaluronan molecule are available for chemical modification: carboxylate, acetamido, the reducing end group, and hydroxyl groups. The bulk of all effort to date has been directed at synthesising medically useful hyaluronan derivatives by using the * Corresponding author. Tel.: q39-0577-232-083; fax q39-0577-232033; e-mail: [email protected]
Fig. 1. Disaccharide unit of Hyal.
hydroxyl and/or the carboxylate group as the point of chemical attack. We modified Hyal by sulphation of the hydroxyl groups using the Py–SO3 complex [2]. By changing the experimental conditions and in particular the sulphating agent/polysaccharide ratio, a series of sulphated hyaluronans (HyalSx) was obtained with a degree of sulphation ranging from 1 SO3y to 4 SO3y groups for a disaccharide unit. The biological activity of HyalSx compounds has been widely evaluated, particularly regarding their heparin-like behaviour [3]. The anticoagulant activity was found to be strictly correlated to their degree of sulphation and to increase with increasing number of sulphated groups. The ability to inactivate thrombin is mainly a direct consequence of the electrostatic interaction with thrombin and does not occur via ATIII as it does for heparin. Moreover, all HyalSx compounds exhibited poor antithrombotic activity, i.e. a modest FXa inactivation was observed [3]. The presence of a large density of negative charges in these macromolecules induces a strong electrostatic repulsion with
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the negatively charged platelet membranes, therefore increasing with the number of sulphated groups. Platelet aggregation is always inhibited by HyalS4 carrying the highest number of negative charges [4].
The platelet activation is connected with specific interaction between the macromolecule and the receptors on the platelet surface. Thus, it is not dependent on the polysaccharide degree of sulphation, but the stimulation of the a-granule
Fig. 2. Structure of the polysaccharide–metal complexes.
Fig. 3. Cross-linking reaction of Hyal.
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is low for all the compounds studied, the lowest being for HyalS3 [4]. The sulphated derivatives of Hyal did not affect the growth of human umbilical vein endothelial cells. The number of cells in medium containing HyalS increased with time. A slightly better growth was observed compared to medium containing Hyal which was similar to that observed for the control. Moreover, endothelial cells in medium containing HyalS spread with no morphological alteration and without structural changes in cell organisation [5]. This behaviour accounts for the angiogenetic effect induced by these macromolecules and particularly by the Cu2q complexes with HyalSx. This is not surprising because the Cu2q heparin complex is also capable of inducing angiogenesis in vitro and in vivo [6]. Indeed, HyalS3.5 can bind several metal ions by its several coordinating groups. The HyalS3.5 macromolecule coordinates Zn2q and Cu2q ions through carboxylate and acetamido moieties without any involvement of the sulphate groups (Fig. 2). Thermodynamic and spectroscopic studies showed the type of complexes formed with Cu2q and Zn2q ions in aqueous solution [7,8]. The complex species found in aqueous solution for all the HyalS3.5–Cu(II) systems as a function of pH are the following: CuL2.5y between pH 3.0 and 6.5, and Cu(OH)2L4.5y at pH)6.0. In the case of HyalS3.5– Zn(II) only one complex species [Zn(OH)2L4.5y] is stable at pH)6.5. As HyalS is water soluble, it is clear that the application of the biological performance of HyalS requires preparations with a longer residence time or greater solidity and elasticity. Further chemical derivatisation of sulphated hyaluronan
Fig. 4. FT-IR-ATR spectra of the Hyal gels at different degrees of crosslinking.
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will provide the opportunity to develop materials for new applications.
3. Hydrogels Several different methods have been used to cross-link hyaluronan [9–12]. By controlling the extent of cross-linking, and the type of covalent bond and the hyaluronan functional group involved, it is possible to create a wide range of physically different materials. Most Hyal polymers are crosslinked via the hydroxyl groups of the hyaluronan chain, leaving the carboxylate and the acetamido groups unreacted. We adopted the strategy to utilise di-amine (such as 1,3-diaminopropan, 1,6-diaminoesan, O,O9-bis(2-aminopropyl)polyethylene glycol 500 and O,O9-bis(2-aminopropyl)polyethylene glycol 500) as cross-linking agents, once the carboxylate groups are activated by 2-chloro-1-methylpyridinium iodite (CMPJ) [13] (Fig. 3). The reaction was performed maintaining the amount of carboxylate activating agent (CMPJ) lower than the number of carboxylate groups present in the weighed quantity of Hyal. Solid-state 13C NMR [14] and FT-IR-ATR (Fig. 4) analysis showed that the quantity of carboxylate groups involved in the cross-linking reaction is regulated by the quantity of CMPJ added to the solution, with the di-amine present in large excess. This allows the physical properties of Hyal to be controlled with-
Fig. 5. Swelling degree (Sw.D.sWw/Wd=100; Wwsweight of wet sample Wdsweight of dry sample) and dynamic elastic modulus (G9) of different Hyal hydrogels.
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Fig. 6. Effect of Cu2q ions on FT-IR spectra of cross-linked Hyal.
Fig. 7. Conductivity experiment in cross-linked HyalS–Cu(II) system.
out unacceptable loss in biocompatibility. The retention of the carboxylate group is especially important because the polyanionic character of Hyal is critical to its physicochem-
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ical and biological properties. We obtain compounds ranging from highly elastoviscous solutions to viscoelastic gels and solids when the cross-linking degree varies from 5 to 100%.
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Article: 6349
R. Barbucci et al. / Journal of Inorganic Biochemistry 79 (2000) 119–125
In addition, the Hyal gels can be sulphated under heterogeneous conditions to improve their haemocompatibility. The rheological analysis indicated a gel-like behaviour of these compounds, being strain independent in small deformation regions. The elastic modulus of the sulphated gels is always higher than that of the non-sulphated ones, indicating that these groups stabilise the gel structure. However, the overall mechanical properties are modulated by the type and length of cross-linking agent. The sulphation reaction did not seem to produce a large effect on the swelling and on the rheological properties (elastic modulus G9) of Hyal crosslinked with the hydrophilic PEG di-amine (Fig. 5). The sulphated gels are able to bind Cu2q ions and the coordinating groups seem similar to those found with the free HyalSx in solution as measured by the FT-IR analysis of the gel before and after the addition of Cu2q. In fact, the IR spectral analysis showed that the introduction of Cu2q induced a shift to lower wavenumber of the peaks assigned to C_O stretching at 1678 and 1660 cmy1 (due to the amide groups of both HyalSx and cross-linking bond), and to the peak at 1625 cmy1 assigned to COOy stretching (Fig. 6). This proves the involvement of all these chemical groups in the Cu2q coordination. The efficacy of these gels to bind Cu2q ions is also well documented by the decrease of conductivity when Cu2q is added to the gel. Fig. 7 shows the conductivity assay of sulphated hydrogel swollen in MOPS (3-(N-morpholino)propansulphonic acid) buffer compared with the MOPS buffer controls [15]. The arrows on the graph show the addition point of the Cu2q solution; the conductivity increase is lower in the hydrogel than in the controls, showing the coordinating ability of hyaluronan, also in cross-linked form. From a biological point of view, the sulphated gels show excellent biocompatibility and a good response to both platelets and primary chondrocytes, which would favour their utilisation in the blood-compatible materials domain and in cartilage repair. The SEM micrograph of Hyal hydrogel after contact with PRP (platelet-rich plasma) shows a very low degree of platelet adhesion. Platelets are also absent inside the mesh of the sample (Fig. 8).
Fig. 8. SEM micrograph of platelet adhesion on Hyal hydrogel. Platelets are also absent inside the meshes of the hydrogel.
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4. Photoimmobilised HyalS In order to immobilise HyalS on a polymer surface, the photoimmobilisation method developed by Matsuda and Sugawara was used [16,17]. As the method does not need the presence of functional groups on the matrix surface to connect the functional unit with the polymer, it has been applied for surface functionalisation of conventional polymers. The photoreactive polymer was obtained by a reaction of HyalS and 4-azidoaniline using water-soluble carbodiimmide (WSC) as activating agent (Fig. 9) [18]. The aqueous solution of the derivatised HyalS was cast onto the polymer substrate poly(ethylene terephthalate) (PET) and irradiated with an UV lamp. The interaction with blood of the HyalS photoimmobilised surface was evaluated in terms of thrombus formation. The amount of thrombus formed in 1 h was about 25% of that formed on the polymer substrate without immobilisation. In
Fig. 9. Preparation of azidophenylamino-derivatised HyalS (AzPhHyalS).
Fig. 10. Photoimmobilisation of AzPhHyalS on PET.
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addition, no thrombus was detected in the initial 20 min. This behaviour corresponds to that of immobilised heparin. Biological performances, in terms of adhesion and proliferation of B10D2 endothelial cells obtained from lung mouse in the HyalS immobilised PET, were compared with the corresponding non-sulphated surface. Results showed that the presence of sulphate groups on the surface is able to induce
Fig. 11. Chondrocytes immunostaining: (a) F-actin on HyalS, (b) CD44 receptor on HyalS, (c) CD44 receptor on PET.
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a better cell proliferation. With the same technique, patterned surfaces were obtained using a photomask between the UV light and the surface containing azido-HyalS (Fig. 10). Strips with different widths were obtained on changing the photomask sizes [18]. These surfaces were used to study the behaviour of B10D2 endothelial cells and primary chondrocytes and an analysis of cell movement on these substrates was performed. Most of the cells migrated from untreated strips to HyalS ones. In particular, F-actin immunostaining of chondrocytes on HyalS showed that the cells were spread and adhered on this substrate. Moreover, CD44 receptor immunostaining appeared more intense in light for cells on HyalS than for the in situ chondrocyte adhesion pathway (Fig. 11).
Fig. 12. Endothelial cells (B10D2) on photoimmobilised HyalS–PET strips 10 mm (a) and 100 mm wide (b). The preference and spreading of cells for HyalS strips was higher in 10 mm strips than in 100 mm ones, demonstrating the role of topographical cues in cell movement.
StyleTag -- Journal: JIB (Journal of Inorganic Biochemistry)
Article: 6349
R. Barbucci et al. / Journal of Inorganic Biochemistry 79 (2000) 119–125
Migration processes of B10D2 endothelial cells were studied on two different HyalS patterned surfaces, 10 and 100 mm wide. The preference and spreading of cells for HyalS strips were higher in 10 mm strips than in 100 mm ones, proving the role of topographical cues in cell movement (Fig. 12).
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ence the migration of primary chondrocytes and B10D2 endothelial cells, so that their utilisation as a scaffold for production of a tissue-engineered cartilage can be hypothesised.
Acknowledgements 5. Conclusions The hyaluronic acid was sulphated and several samples with different sulphation degrees were obtained. The sulphated groups provided the Hyal macromolecule with peculiar characteristics in terms of polyelectrolyte behaviour: the HyalS macromolecule remained in the stretched conformation at any pH value of the solution, whereas the Hyal coiled once the carboxyl groups were protonated (pH-4.0). The insertion of sulphate groups also provided the macromolecule with anticoagulant properties, resistance to enzymatic digestion, and ability to inhibit the platelet adhesion and aggregation. HyalS derivatives were able to form complexes with Cu(II) and Zn(II) ions and these complexes, in particular the Cu(II)–HyalS complex, were demonstrated to favour endothelial cell adhesion and migration. Considering the good biological properties of these modified macromolecules, new biomaterials were synthesised using HyalS as one of the components. Hydrogels with different physicochemical and mechanical properties were obtained by cross-linking the Hyal chains. The ability of the sulphated gels to complex Cu2q ions was demonstrated and the same coordinating groups as those of the free HyalS seemed to be involved in the complex formation. The sulphated gels showed an excellent biocompatibility and a good response to both platelet and primary chondrocytes, so that their utilisation in the blood-compatible materials domain in cartilage repair can be envisaged. Micropatterned surfaces with improved thromboresistance were obtained by photoimmobilisation of HyalS on PET substrates. The 10 m micropatterned surfaces were able to influ-
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We thank the ‘Progetto Finalizzato MSTA II’ of the National Research Council for financial support and the ‘Center for Cell Engineering’ (University of Glasgow, UK) for cell movement and conductivity studies.
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StyleTag -- Journal: JIB (Journal of Inorganic Biochemistry)
Article: 6349