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PVDF multifilament yarns grafted with polystyrene induced by γ-irradition: Influence of the grafting parameters on the mechanical properties
Nuclear Instruments and Methods in Physics Research B 208 (2003) 429–433 www.elsevier.com/locate/nimb
PVDF multifilament yarns grafted with polystyrene induced by c-irradition: Influence of the grafting parameters on the mechanical properties P. Marmey *, M.C. Porte, Ch. Baquey U577 INSERM, Universit e Victor Segalen Bordeaux 2, 146 rue L eo Saignat, 33076 Bordeaux cedex, France
Abstract The development of alternative prosthetic materials for cardiovascular applications has found growing interest due to the failure to date to be able to implement functional patent small diameter vascular grafts (£ < 5 mm). For instance, the successful implantation of small diameter polyester (PET) and expanded polytetrafluoroethylene (PTFEe) vascular grafts has not been achieved in humans. Our stategy is to work with a new multifilament yarns biomaterial, produced from polyvinylidene fluoride (PVDF), which shows suitable mechanical properties, such as a lower tensile modulus than PET and PTFEe. The required biological properties sought for (i.e. low thrombogenicity) could be achieved by ‘‘heparin-like’’ surface modification treatments in order to modify the thrombogenicity levels of the polymeric materials [Ann. Biomed. Eng. 7 (1979) 429]. A four step method is necessary to achieve this ‘‘heparin-like’’ surface transformation [J. Biomed. Mater. Res. 52 (2000) 119]. The first step consists in grafting polystyrene onto the PVDF surface by c irradiation. The purpose of this study was to evaluate the influence of grafting parameters on the mechanical properties: (i) c-ray irradiation time and (ii) grafting time of styrene monomers, which polymerize and form polystyrene bound to the PVDF surface. Ó 2003 Elsevier B.V. All rights reserved. PACS: 07.85.)m; 81.05.Lg; 87.15.La; 87.68.+z Keywords: Poly(vinylidene fluoride); Heparin-like; c-rays; Polystyrene grafting
1. Introduction The successful substitution of small diameter arteries (£ < 5 mm) by polyester (PET) and expanded teflon (polytetrafluoroethylene, PTFEe)
* Corresponding author. Tel.: +33-5-57-57-14-88; fax: +33-556-90-05-17. E-mail address: [email protected] (P. Marmey).
vascular grafts has not been achieved in humans. To this end, we have chosen to work with new multifilament yarns extruded from polyvinylidene fluoride (PVDF) which present suitable mechanical properties, i.e. lower tensile modulus than PET and PTFE, and has been subjected to a special treatment called ‘‘heparin-like’’. Sulfonate and sulfonamide groups (sulfonamide of aspartic acid) were introduced on phenyl rings belonging to styrene residues which were radiation grafted (gamma radiation) onto PVDF. These groups,
0168-583X/03/$ - see front matter Ó 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0168-583X(03)00887-5
430
P. Marmey et al. / Nucl. Instr. and Meth. in Phys. Res. B 208 (2003) 429–433
known to be responsible for a specific affinity for Antithrombin III (ATIII) and thus able to catalyse the inhibition of thrombin by ATIII, as heparin does [1,3], and lower the thrombogenicity. The purpose of this study was to evaluate the mechanical properties of the PS-PVDF grafted copolymers, with respect to synthesis parameters and to study the effect of the latter on the former. A series of mechanical tests were performed to investigate the tensile properties such as YoungÕs modulus and energy to break. Fig. 2. Second step – styrene grafting.
2. Materials and methods 2.1. Materials PVDF SOLEFâ multifilament (42 filaments) yarns with a linear density s ¼ 601 dTex (g/10,000 m) were provided from the Polisilk SA company (Manresa, Barcelone, Espagne). Each filament had a 32.8 lm diameter and a linear density of sf ¼ 15 dTex. Preliminary results concerning development of ‘‘heparin-like’’ PVDF films had already been obtained [2,4]. In the same way as for the films, the surface of PVDF filaments was first grafted with polystyrene after gamma irradiation under normal atmosphere (dose rate: 2.5 kGy h1 ) and subsequently treated in order to acquire heparin-like properties. A four step procedure was performed [5]. This technique is described in details elsewhere [4,6] and the two first steps consisting of a gamma irradiation followed by a PS grafting are detailed below: Polystyrene was ‘‘grafted from’’ the PVDF yarns surface, after gamma irradiation (Fig. 1), by radical polymerisation of styrene (Fig. 2) (on it).
The mass of covalently bound polystyrene on the PVDF depends upon the c-ray dose D (kGy or kilograys) and styrene grafting time t (h). The following equation was used to calculate the grafting yield, Y (%): mPS Y ð%Þ ¼ : mPVDF 2.2. Tensile behaviour Measurements of the stress–strain behaviour (percent – kN/tex) of nine PVDF yarns were investigated using out on an Instron universal tester, model 4466, fitted with a 500 N load cell. A crosshead speed of 500 mm/min was used to extend specimens from an initial gauge length of 100 mm. The YoungÕs modulus in kilonewtons per Tex (kN/ Tex) and the energy to break in kilojoules per Tex (kJ/Tex) were calculated and averaged from recorded stress–strain curves, knowing that each sample was tested four times. The yarn size used in the stress measurement, is the real filament diameter, measured with an optical microscope (Nikon Optiphot 2) at 40 magnification equipped with an image analyzer (Biocom visiol@b) and a calibrated eyepiece micrometer. 3. Results and discussion 3.1. Tensile behaviour
Fig. 1. PVDF c-irradiation mechanism, creating stable peroxides.
In order to measure the influence of each step of the polystyrene grafting on the yarns, tensile tests
P. Marmey et al. / Nucl. Instr. and Meth. in Phys. Res. B 208 (2003) 429–433
Irradiation did not change the PVDF yarns YoungÕs modulus. This result confirms the good behaviour of the PVDF under irradiation [7], especially in the elastic strain zone. Energy to break results are reported on Fig. 5. From 0 to 2.7 kGy, no influence was observed. From 2.7 up to 8.1 kGy an important increase of the Energy to break was explained by a high crosslinking yield of the PVDF due to the irradiation at low doses [8]. Then a regular decrease down to a very weak value of the energy was observed from 8.1 kGy to the highest dose (54 kGy). This last value is twice lower than the initial value. This drop is probably due to a great chain scissions yield [8].
Fig. 3. Typical strain/stress curve.
were carried out for (i) the c-irradiated yarns (Fig. 1) and for (ii) the PS grafted PVDF yarns (Fig. 2). Two mechanical parameters were calculated:
3.1.2. Influence of the grafting parameters Table 1 presents grafting parameters (dose, grafting time) used to graft PS on PVDF surfaces. YoungÕs modulus data related to the materials reported in Table 1 are reported on Fig. 6. The YoungÕs modulus increases with the dose D, the grafting time t and consequently the grafting yield Y . That could be explained by the large quantities of grafted PS on the PVDF surface, which is a more rigid polymer [9]. Energy to break data are reported on Fig. 7 For doses greater than 2.7 kGy, this treatment has a bad influence on the energy to break. A 20% reduction of this energy is observed with a 2.7 kGy dose. A 54 kGy dose induces a 300% degradation.
1. YoungÕs modulus (kN/Tex). Initial slope of the strain/stress curves. 2. Energy to break (kJ/Tex), indirectly calculated from the area under the strain/stress curves (Fig. 3).
kN/Tex
3.1.1. Influence of the radiation dose Tensile properties of five virgin PVDF yarns c-irradiated with absorbed doses ranging from 2.7 to 8.1 kGy were tested. YoungÕs modulus results are reported on Fig. 4.
1.2E-03
1.0E-03
8.0E-04
6.0E-04
4.0E-04
2.0E-04
0.0E+00 PVDF control
431
2.7
8.1
24.3
54.0 81.0 Doses kGy
Fig. 4. YoungÕs modulus of five yarns c-irradiated with doses ranging from 2.7 to 81 kGy.
P. Marmey et al. / Nucl. Instr. and Meth. in Phys. Res. B 208 (2003) 429–433 kJ/Tex
432
1.2E-04
1.0E-04
8.0E-05
6.0E-05
4.0E-05
2.0E-05
0.0E+00 PVDF control
2.7
8.1
24.3
54.0
Dose kGy
81.0
Fig. 5. Energy to break of five yarns c-irradiated with doses ranging from 2.7 to 81 kGy.
Table 1 Characteristics of eight materials studied with tensile tests Dose D (kGy)
Grafting time t (h)
Grafting yield Y (%)
Linear density s (dTex)
Virgin PVDF 2.7 kGy + 1 h 2.7 kGy + 2 h 8.1 kGy + 1 h 8.1 kGy + 2 h 24.3 kGy + 1 h 24.3 kGy + 2 h 54 kGy + 1 h 54 kGy + 2 h
– 2.7 2.7 8.1 8.1 24.3 24.3 54 54
– 1 2 1 2 1 2 1 2
– 17.4% 23.6% 30.5% 31.6% 35.1% 43.6% 33.7% 51.0%
601 715 650 733 760 899 883 711 860
1.6E-03 1.4E-03 1.2E-03 Control
1.0E-03 8.0E-04 6.0E-04 4.0E-04 2.0E-04
54 kG y+ 2h
54 kG y+ 1h
24 ,3 kG y+ 1h 24 ,3 kG y+ 2h
8, 1k G y+ 2h
8, 1k G y+ 1h
2, 7k G y+ 2h
F D
2, 7k G y+ 1h
0.0E+00 PV
Young Modulus (kN/tex)
Materials
Dose kGy + Grafting time t
Fig. 6. YoungÕs modulus of eight different grafted materials.
Fracture Toughness (kJ/tex)
P. Marmey et al. / Nucl. Instr. and Meth. in Phys. Res. B 208 (2003) 429–433
433
9.0E-05 8.0E-05 7.0E-05
Control
6.0E-05 5.0E-05 4.0E-05 3.0E-05 2.0E-05 1.0E-05
2h 54
kG
y+ kG
y+
1h
2h 54
24
,3
kG
y+ 24
,3
kG
G 1k 8,
y+
1h
2h y+
1h y+ 8,
1k
G
y+ G 7k 2,
2,
7k
G
PV
y+
D
F
1h
2h
0.0E+00
Dose kGy + Grafting time t
Fig. 7. Energy to break of eight different grafted materials.
The chain scissions in the PVDF for large irradiation doses as shown by Fig. 7 and the strong increase of the PS content in the filament, which is a brittle material, explain this phenomenon.
the INSERM and the ‘‘Laboratoire de Chimie des Polymeres Organiques’’ LCPO – Bordeaux – France, for their technical help.
4. Conclusions
References
We have performed radiation grafting of PS on PVDF yarns. The influence of the synthesis parameters, (i) c-rays dose D (kGy) and (ii) grafting time t (h) is well-established. The bulk properties of the materials were analysed by tensile tests. 8.1 kGy is a high enough dose to initiate significant changes on energy to break, without any change on the YoungÕs modulus. Limiting the absorbed dose (D < 8:1 kGy) and the grafting time (1 < t < 2 h) is necessary to limit any deleterious effect on the PS grafted PVDF yarns tensile properties.
[1] C. Fougnot, J. Jozefonvicz, M. Samama, L. Bara, Ann. Biomed. Eng. 7 (1979) 429. [2] M.C. Porte-Durrieu, C. Aymes-Chodur, N. Betz, C. Baquey, J. Biomed. Mater. Res. 52 (2000) 119. [3] C. Fougnot, J. Jozefonvicz, M. Samama, L. Bara, Ann. Biomed. Eng. 7 (1979-2) 441. [4] M.C. Porte-Durrieu, C. Aymes-Chodur, N. Betz, B. Brouillaud, F. Rouais, A. Le Moel, C. Baquey, Nucl. Instr. and Meth. B 131 (1997) 364. [5] V. Migonney, C. Fougnot, M. Jozefowicz, Biomaterials 9 (1988-1) 145. [6] M.C. Porte-Durrieu, C. Aymes-Chodur, C. Vergne, N. Betz, C. Baquey, Nucl. Instr. and Meth. B 151 (1999) 404. [7] J. Scheirs, in: Modern Fluoropolymers: High Performance Polymers for Diverse Applications, Vol. 1, Wiley, New York, 1997, p. 335. [8] N. Betz, E. Petersohn, A. Le Moel, Nucl. Instr. and Meth. B 116 (1996) 207. [9] J.I. Kroschwitz, Concise Encyclopedia of Polymer Science and Engineering, Wiley, Chichester, New York, 1998, p. 1117.
Acknowledgements The authors gratefully acknowledge the financial support of the French Research Minister and
PVDF multifilament yarns grafted with polystyrene induced by c-irradition: Influence of the grafting parameters on the mechanical properties P. Marmey *, M.C. Porte, Ch. Baquey U577 INSERM, Universit e Victor Segalen Bordeaux 2, 146 rue L eo Saignat, 33076 Bordeaux cedex, France
Abstract The development of alternative prosthetic materials for cardiovascular applications has found growing interest due to the failure to date to be able to implement functional patent small diameter vascular grafts (£ < 5 mm). For instance, the successful implantation of small diameter polyester (PET) and expanded polytetrafluoroethylene (PTFEe) vascular grafts has not been achieved in humans. Our stategy is to work with a new multifilament yarns biomaterial, produced from polyvinylidene fluoride (PVDF), which shows suitable mechanical properties, such as a lower tensile modulus than PET and PTFEe. The required biological properties sought for (i.e. low thrombogenicity) could be achieved by ‘‘heparin-like’’ surface modification treatments in order to modify the thrombogenicity levels of the polymeric materials [Ann. Biomed. Eng. 7 (1979) 429]. A four step method is necessary to achieve this ‘‘heparin-like’’ surface transformation [J. Biomed. Mater. Res. 52 (2000) 119]. The first step consists in grafting polystyrene onto the PVDF surface by c irradiation. The purpose of this study was to evaluate the influence of grafting parameters on the mechanical properties: (i) c-ray irradiation time and (ii) grafting time of styrene monomers, which polymerize and form polystyrene bound to the PVDF surface. Ó 2003 Elsevier B.V. All rights reserved. PACS: 07.85.)m; 81.05.Lg; 87.15.La; 87.68.+z Keywords: Poly(vinylidene fluoride); Heparin-like; c-rays; Polystyrene grafting
1. Introduction The successful substitution of small diameter arteries (£ < 5 mm) by polyester (PET) and expanded teflon (polytetrafluoroethylene, PTFEe)
* Corresponding author. Tel.: +33-5-57-57-14-88; fax: +33-556-90-05-17. E-mail address: [email protected] (P. Marmey).
vascular grafts has not been achieved in humans. To this end, we have chosen to work with new multifilament yarns extruded from polyvinylidene fluoride (PVDF) which present suitable mechanical properties, i.e. lower tensile modulus than PET and PTFE, and has been subjected to a special treatment called ‘‘heparin-like’’. Sulfonate and sulfonamide groups (sulfonamide of aspartic acid) were introduced on phenyl rings belonging to styrene residues which were radiation grafted (gamma radiation) onto PVDF. These groups,
0168-583X/03/$ - see front matter Ó 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0168-583X(03)00887-5
430
P. Marmey et al. / Nucl. Instr. and Meth. in Phys. Res. B 208 (2003) 429–433
known to be responsible for a specific affinity for Antithrombin III (ATIII) and thus able to catalyse the inhibition of thrombin by ATIII, as heparin does [1,3], and lower the thrombogenicity. The purpose of this study was to evaluate the mechanical properties of the PS-PVDF grafted copolymers, with respect to synthesis parameters and to study the effect of the latter on the former. A series of mechanical tests were performed to investigate the tensile properties such as YoungÕs modulus and energy to break. Fig. 2. Second step – styrene grafting.
2. Materials and methods 2.1. Materials PVDF SOLEFâ multifilament (42 filaments) yarns with a linear density s ¼ 601 dTex (g/10,000 m) were provided from the Polisilk SA company (Manresa, Barcelone, Espagne). Each filament had a 32.8 lm diameter and a linear density of sf ¼ 15 dTex. Preliminary results concerning development of ‘‘heparin-like’’ PVDF films had already been obtained [2,4]. In the same way as for the films, the surface of PVDF filaments was first grafted with polystyrene after gamma irradiation under normal atmosphere (dose rate: 2.5 kGy h1 ) and subsequently treated in order to acquire heparin-like properties. A four step procedure was performed [5]. This technique is described in details elsewhere [4,6] and the two first steps consisting of a gamma irradiation followed by a PS grafting are detailed below: Polystyrene was ‘‘grafted from’’ the PVDF yarns surface, after gamma irradiation (Fig. 1), by radical polymerisation of styrene (Fig. 2) (on it).
The mass of covalently bound polystyrene on the PVDF depends upon the c-ray dose D (kGy or kilograys) and styrene grafting time t (h). The following equation was used to calculate the grafting yield, Y (%): mPS Y ð%Þ ¼ : mPVDF 2.2. Tensile behaviour Measurements of the stress–strain behaviour (percent – kN/tex) of nine PVDF yarns were investigated using out on an Instron universal tester, model 4466, fitted with a 500 N load cell. A crosshead speed of 500 mm/min was used to extend specimens from an initial gauge length of 100 mm. The YoungÕs modulus in kilonewtons per Tex (kN/ Tex) and the energy to break in kilojoules per Tex (kJ/Tex) were calculated and averaged from recorded stress–strain curves, knowing that each sample was tested four times. The yarn size used in the stress measurement, is the real filament diameter, measured with an optical microscope (Nikon Optiphot 2) at 40 magnification equipped with an image analyzer (Biocom visiol@b) and a calibrated eyepiece micrometer. 3. Results and discussion 3.1. Tensile behaviour
Fig. 1. PVDF c-irradiation mechanism, creating stable peroxides.
In order to measure the influence of each step of the polystyrene grafting on the yarns, tensile tests
P. Marmey et al. / Nucl. Instr. and Meth. in Phys. Res. B 208 (2003) 429–433
Irradiation did not change the PVDF yarns YoungÕs modulus. This result confirms the good behaviour of the PVDF under irradiation [7], especially in the elastic strain zone. Energy to break results are reported on Fig. 5. From 0 to 2.7 kGy, no influence was observed. From 2.7 up to 8.1 kGy an important increase of the Energy to break was explained by a high crosslinking yield of the PVDF due to the irradiation at low doses [8]. Then a regular decrease down to a very weak value of the energy was observed from 8.1 kGy to the highest dose (54 kGy). This last value is twice lower than the initial value. This drop is probably due to a great chain scissions yield [8].
Fig. 3. Typical strain/stress curve.
were carried out for (i) the c-irradiated yarns (Fig. 1) and for (ii) the PS grafted PVDF yarns (Fig. 2). Two mechanical parameters were calculated:
3.1.2. Influence of the grafting parameters Table 1 presents grafting parameters (dose, grafting time) used to graft PS on PVDF surfaces. YoungÕs modulus data related to the materials reported in Table 1 are reported on Fig. 6. The YoungÕs modulus increases with the dose D, the grafting time t and consequently the grafting yield Y . That could be explained by the large quantities of grafted PS on the PVDF surface, which is a more rigid polymer [9]. Energy to break data are reported on Fig. 7 For doses greater than 2.7 kGy, this treatment has a bad influence on the energy to break. A 20% reduction of this energy is observed with a 2.7 kGy dose. A 54 kGy dose induces a 300% degradation.
1. YoungÕs modulus (kN/Tex). Initial slope of the strain/stress curves. 2. Energy to break (kJ/Tex), indirectly calculated from the area under the strain/stress curves (Fig. 3).
kN/Tex
3.1.1. Influence of the radiation dose Tensile properties of five virgin PVDF yarns c-irradiated with absorbed doses ranging from 2.7 to 8.1 kGy were tested. YoungÕs modulus results are reported on Fig. 4.
1.2E-03
1.0E-03
8.0E-04
6.0E-04
4.0E-04
2.0E-04
0.0E+00 PVDF control
431
2.7
8.1
24.3
54.0 81.0 Doses kGy
Fig. 4. YoungÕs modulus of five yarns c-irradiated with doses ranging from 2.7 to 81 kGy.
P. Marmey et al. / Nucl. Instr. and Meth. in Phys. Res. B 208 (2003) 429–433 kJ/Tex
432
1.2E-04
1.0E-04
8.0E-05
6.0E-05
4.0E-05
2.0E-05
0.0E+00 PVDF control
2.7
8.1
24.3
54.0
Dose kGy
81.0
Fig. 5. Energy to break of five yarns c-irradiated with doses ranging from 2.7 to 81 kGy.
Table 1 Characteristics of eight materials studied with tensile tests Dose D (kGy)
Grafting time t (h)
Grafting yield Y (%)
Linear density s (dTex)
Virgin PVDF 2.7 kGy + 1 h 2.7 kGy + 2 h 8.1 kGy + 1 h 8.1 kGy + 2 h 24.3 kGy + 1 h 24.3 kGy + 2 h 54 kGy + 1 h 54 kGy + 2 h
– 2.7 2.7 8.1 8.1 24.3 24.3 54 54
– 1 2 1 2 1 2 1 2
– 17.4% 23.6% 30.5% 31.6% 35.1% 43.6% 33.7% 51.0%
601 715 650 733 760 899 883 711 860
1.6E-03 1.4E-03 1.2E-03 Control
1.0E-03 8.0E-04 6.0E-04 4.0E-04 2.0E-04
54 kG y+ 2h
54 kG y+ 1h
24 ,3 kG y+ 1h 24 ,3 kG y+ 2h
8, 1k G y+ 2h
8, 1k G y+ 1h
2, 7k G y+ 2h
F D
2, 7k G y+ 1h
0.0E+00 PV
Young Modulus (kN/tex)
Materials
Dose kGy + Grafting time t
Fig. 6. YoungÕs modulus of eight different grafted materials.
Fracture Toughness (kJ/tex)
P. Marmey et al. / Nucl. Instr. and Meth. in Phys. Res. B 208 (2003) 429–433
433
9.0E-05 8.0E-05 7.0E-05
Control
6.0E-05 5.0E-05 4.0E-05 3.0E-05 2.0E-05 1.0E-05
2h 54
kG
y+ kG
y+
1h
2h 54
24
,3
kG
y+ 24
,3
kG
G 1k 8,
y+
1h
2h y+
1h y+ 8,
1k
G
y+ G 7k 2,
2,
7k
G
PV
y+
D
F
1h
2h
0.0E+00
Dose kGy + Grafting time t
Fig. 7. Energy to break of eight different grafted materials.
The chain scissions in the PVDF for large irradiation doses as shown by Fig. 7 and the strong increase of the PS content in the filament, which is a brittle material, explain this phenomenon.
the INSERM and the ‘‘Laboratoire de Chimie des Polymeres Organiques’’ LCPO – Bordeaux – France, for their technical help.
4. Conclusions
References
We have performed radiation grafting of PS on PVDF yarns. The influence of the synthesis parameters, (i) c-rays dose D (kGy) and (ii) grafting time t (h) is well-established. The bulk properties of the materials were analysed by tensile tests. 8.1 kGy is a high enough dose to initiate significant changes on energy to break, without any change on the YoungÕs modulus. Limiting the absorbed dose (D < 8:1 kGy) and the grafting time (1 < t < 2 h) is necessary to limit any deleterious effect on the PS grafted PVDF yarns tensile properties.
[1] C. Fougnot, J. Jozefonvicz, M. Samama, L. Bara, Ann. Biomed. Eng. 7 (1979) 429. [2] M.C. Porte-Durrieu, C. Aymes-Chodur, N. Betz, C. Baquey, J. Biomed. Mater. Res. 52 (2000) 119. [3] C. Fougnot, J. Jozefonvicz, M. Samama, L. Bara, Ann. Biomed. Eng. 7 (1979-2) 441. [4] M.C. Porte-Durrieu, C. Aymes-Chodur, N. Betz, B. Brouillaud, F. Rouais, A. Le Moel, C. Baquey, Nucl. Instr. and Meth. B 131 (1997) 364. [5] V. Migonney, C. Fougnot, M. Jozefowicz, Biomaterials 9 (1988-1) 145. [6] M.C. Porte-Durrieu, C. Aymes-Chodur, C. Vergne, N. Betz, C. Baquey, Nucl. Instr. and Meth. B 151 (1999) 404. [7] J. Scheirs, in: Modern Fluoropolymers: High Performance Polymers for Diverse Applications, Vol. 1, Wiley, New York, 1997, p. 335. [8] N. Betz, E. Petersohn, A. Le Moel, Nucl. Instr. and Meth. B 116 (1996) 207. [9] J.I. Kroschwitz, Concise Encyclopedia of Polymer Science and Engineering, Wiley, Chichester, New York, 1998, p. 1117.
Acknowledgements The authors gratefully acknowledge the financial support of the French Research Minister and