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EVOH biomaterials
Polymer Degradation and Stability 73 (2001) 237–244 www.elsevier.nl/locate/polydegstab
In-vitro degradation behaviour of starch/EVOH biomaterials M. Alberta Arau´jo a, Cla´udia M. Vaz b, Anto´nio M. Cunha b, Manuel Mota a,* a
Centro de Engenharia Biolo´gica (IBQF), Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal Department of Polymer Engineering, Universidade do Minho, Campus de Azure´m, 4800-058 Guimara˜es, Portugal
b
Received 28 June 2000; received in revised form 8 January 2001; accepted 17 January 2001
Abstract Blends of corn starch with poly(ethylene–vinyl alcohol) copolymer (SEVA-C) were aged up to 120 days on NaCl sterile isotonic solution (9 g/l) and the degradation solutions were analysed by several techniques. Mechanical properties (i.e. strength, stiffness and ductility), weight loss, and water uptake were also assessed. The evolution of the material structure was followed by scanning electron microscopy (SEM), contact angle measurements and thermogravimetric analysis (TGA). The degradation solutions were monitored with anionic chromatography (HPAE-PAD), colorimetric methods, and Fourier transformed infra-red spectrophotomety (FT-IR) in order to quantify the amount of plasticisers and sugars. SEVA-C micrographs presented an increase in the surface porosity as a function of immersion time. The main compound released to the solution is glycerol, the other degradation products are low molecular weight polymeric chains. From the physical–chemical point of view the material degradation is principally associated with the leaching of glycerol, since the other metabolites were not released. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Biodegradable polymer; In-vitro degradation; Biomedical; Starch/poly(ethylene–vinyl alcohol)
1. Introduction Bioabsorbable polymers may be advantageous for orthopaedic fracture fixation devices, mainly if they present an appropriate combination of initial strength and stiffness with a controlled degradation rate. The expectable reduction in the modulus of the biomaterial should lead to a gradual load transfer to the healing bone, and eventually, the complete absorption of the implant may avoid a second operation to its removal [1–3]. Systems based on poly(lactic acid) (PLA) [1,2], poly (glycolic acid) (PGA) [1] and their copolymers and poly(hydroxybutyrate) (PHB) [1,2] are well known examples of biodegradable polymers in orthopaedics. Blends of native corn starch with poly(ethylene–vinyl alcohol) copolymer (SEVA-C), are potential alternatives to the referred polymers used in clinical applications [4–6]. This class of materials has been reported as being biodegradable [7–11] but only recently were proposed for possible use as biomaterials [4–6]. The great advantage of this kind of materials as compared with the synthetic ones, is their * Corresponding author. Tel.: +351-253-604405; fax: +351-253678986. E-mail address: [email protected] (M. Mota).
biocompatibility with the organic tissues and their similarity with the bone dynamic mechanical behaviour. Certain performance requirements must be imposed upon materials intended to be used in contact with living systems [12]. Many of these specifications naturally fall into two categories: the effect of the organism on the implant and the effect of the implant on the organism [13]. These minimum standards include numerous criteria such as: (i) the material must not leach or release soluble components into the living systems, unless this release is intentional and/or non-toxic; (ii) the living system must not degrade the implant, unless this degradation is intentional [13]; (iii) the mechanical and physical behaviour of the polymer must be appropriate for the intended function and the desired mechanical properties must persist during the expected life of the implant; and finally (iv) the materials must be biocompatible, sterilisable and free of adherent bacteria and endotoxins from the bacteria cell walls [13]. In general, a polymeric material must be specifically engineered to meet these stringent demands [13] being the polymer degradability one of the most important factors leading to the acceptability of polymers for medical use [15]. Degradation, which can be defined as a damage on the chemical structure or a loss on physical
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properties or appearance of a plastic compound [14], can include [13]: polymeric chain cleavage (reduction in molecular weight), leaching of plasticisers and additives, chain crosslinking (increase in effective molecular weight), side chain hydrolysis or reaction and inappropriate swelling (from water or lipids). The in-vitro and in-vivo degradation of polymeric systems can be affected by various factors such as: polymer chemical structure and molecular mass, formulation and morphology, and thickness of the used specimen [16]. In most situations, degradation is undesirable since the device will be intended to remain in the body for a long period of time without any change in its functional properties and without inducing any significant adverse response from the tissues [15]. In other cases, however, degradation may be intentional, if either the function is transient so that the device requires elimination at an appropriate time, or if the function relies upon some degenerative process, as in drug or other biologically active agent release [17,18]. The degradation behaviour of insoluble polymers depends on both chemical structure and physical state, being of major importance the hydrophilicity (as in the case of starch based materials) [19,20]. Since the aqueous environment of the body remains reasonably constant (pH=7.4 and T=37 C) it should be possible to predict the susceptibility and kinetics of the in-vivo degradation process, considering hydrolysis the only mechanism present [15]. The in-vitro degradation tests, in physiological isotonic solutions, are able to simulate the interactions between the body fluids and a biomaterial, and can allow for the understanding of its stability and degradation rate. Despite being impossible to completely simulate the chemical, mechanical and dynamic character of the in-vivo system in order to predict the behaviour of biomaterials in the human physiological environment, in-vitro and in-vivo studies form an integral part of testing potential implant materials. Although biodegradable polymers are preferentially composed of residues normally present in the human body, implants made of these materials often trigger inflammatory responses. This paper presents a preliminary approach to the study of the mechanisms involved in the in-vitro degradation of a biomaterial based on starch/poly(ethylene–vinyl alcohol) blend, known as an alternative to the polymers currently used in clinical applications.
2. Materials and methods 2.1. Materials The material studied was a thermoplastic blend of corn starch with a poly(ethylene–vinyl alcohol) copolymer (60/40 mol/mol), SEVA-C, supplied by Novamont,
Novara, Italy. Two different types of specimens (tensile bars and square plates, with cross sections of 24 and 230 mm, respectively) were moulded in a Klockner Ferromatic FM-20 injection machine. The SEVA-C samples, were weighed and immersed for several pre-fixed ageing periods (until 120 days) in a sterile isotonic solution (NaCl 9 g/l) at 37 C and pH=7.4. The tensile bars were used to evaluate the material water uptake and weight loss and also for mechanical testing. The plates were used in in-vitro degradation experiments under strictly controlled conditions in a laminar flux chamber. The samples were sterilized by autoclave in an atmosphere of 12/88 mixture of ethylene oxide (EtO) and carbon dioxide (CO2), with a cycle time of 14 h at a working temperature of 45 C, a moisture level of 50% and a chamber pressure of 50 kPa. 2.2. Physical and mechanical characterization After being removed from the degradation solutions (50 ml), the tensile bars were immediately weighed to evaluate the respective water uptake. One batch (three specimens of about 1.300 g each) was dried up to exhaustion in an oven for 72 h at 70 2 C in order to determine the respective weight loss. Another batch was conditioned in a chamber with a controlled atmosphere (23 C and 55% RH) for 1 week, to stabilise the moisture content. In order to evaluate the changes in the mechanical properties as a function of the immersion time, the specimens from the conditioned batch were tested in an Instron 4505 universal machine, fitted with a resistive extensometer (gauge length 10 mm), in a controlled environment (23 C and 55% RH). The crosshead velocity was 8.310 5 m/s (5 mm/min) until 1% strain and then increased to 8.310 4 m/s (50 mm/min) until fracture. These tests were aimed at determining the ultimate tensile strength (UTS), the secant modulus at 1% strain (E1%) and the strain at break ("r). 2.3. Analytical and morphologycal methods 2.3.1. Thermogravimetric analysis Thermogravimetric analysis (TGA) was performed in a TGA-50 Shimadzu-thermogravimetric equipment using aluminium pans, under the following conditions: heating rate of 5 C/min, hold temperature and time of 120 C and 110 min, respectively, and a constant helium flow of 30 ml/min. Samples of about 18 mg were used for the TGA tests. The samples were removed for testing at pre-defined immersion time (until 120 days) and kept in a exsicator, with controlled temperature and humidity, until constant weight. All the assays were performed in triplicate, considering the medium of the three results as the final result.
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2.3.2. Detection of starch and polysaccharides The starch amount in the degradation solutions was determined by means of a simple colorimetric method. 300 ml of sulphuric acid 2 M and 0.5 ml of KI-I2 were added to a 5 ml of sample of the degradation solution. The absorbance of the resulting solution was determined at a wavelength of 580 nm, being the respective concentration obtained from a standard curve, obtained with the same corn starch used in the samples. The total amount of polysaccharides in the degradation solutions was quantified using the Dubois method [21] which is based on the addition of 1 ml of phenol (5% w/v) and 5 ml sulphuric acid (95–97%) to 1 ml of sample of the degradation solution. The absorbance of the resulting mixture was determined at a wavelength of 490 nm using water in the reference cell. 2.3.3. Detection of saccharides and glycerol with high performance anion exchange chromatography High performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD) equipped with a Carbopac PA1 (4250 mm) column and a Carbopac guard column (450 mm) was used to separate saccharides and glycerol from the degradation solutions. Separation was performed with a flow rate of 1 ml/ min using an eluent gradient of 50% of NaOH 0.2 M and ultra-pure water (referred as the 50% gradient). Eluents were flushed with helium to remove dissolved gases and were continuously pressurised with the eluent degas module of Dionex. Another gradient of 20% NaOH and 80% water was also used to elute the monosaccharides and disaccharides after the Soxtec extraction described below. This gradient was applied to separate glycerol and saccharides from all the degradation solutions at different times. Lactose (50 mg/l) was used as the internal standard. 2.3.4. Soxtec extraction Total extraction was used to isolate soluble matter, such as additives, from the studied material. The extraction was performed in a Soxtec system HT 2 Tecator during 12 h. SEVA-C square specimens were weighed into thimbles and inserted into the extraction unit. After solvent addition (water was used as the extraction medium) to the extraction cups, the soluble material was extracted in a two stage process. First, the sample was immersed in boiling water to dissolve most of the soluble material. In the second step, the sample was raised above the solution to enable an efficient washing with water from the condensers. Finally, the extraction cups were dried and weighed. The obtained extracted solutions were injected in HPAE-PAD using the 50% gradient described above to analyse the glycerol and saccharide content.
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2.3.5. FTIR Degradation solutions, of different ageing periods, were freeze dried in a Freeze Dryer Alpha 2-4. The drying was achieved by avoiding the liquid state through sublimation using the following procedure: (i) pre-freezing the degradation solutions; (ii) freeze-drying (24 h) according to a pre-selected temperature ( 70 C) and vacuum level (0.05 mbar); and (iii) final drying of products to remove the capillary and molecular bound water. After freeze drying, the resulting powders were conditioned in an exsicator before the preparation in KBr pellets for FTIR analysis. FTIR assays were performed in a Perkin-Elmer FT-IR 1600 spectrophotometer. 2.3.6. Contact angle measurements The plates were dried at 80 C for 1 week and kept in a exsicator. Contact angle measurements were carried out at room temperature using the sessile drop technique, with a video camera mounted on a microscope to record the drop image. Water was used for this purpose. Sessile drops were deposited with a micrometric syringe directly on the surface from that metallic needle. Pictures of the drops were taken, at regular intervals in a standard contact angle apparatus. At least 30 drops of about 6 ml for each sample were analysed. 2.3.7. SEM The effect of the solution on the surface morphology of the SEVA-C specimens, as a function of immersion time, was followed by scanning electron microscopy (SEM) in a Leica Cambridge S360 microscope. The plates were dried at 80 C for 1 week and kept in a exsicator before the analysis. For control, a non-immersed specimen, stored at controlled environment conditions was used.
3. Results 3.1. Thermogravimetric behaviour and surface morphology of SEVA-C The change of the material surface with the immersion time was morphologically assessed by SEM. The SEVA-C surfaces micrographs (Figs. 1–3) showed the change of the material surface as a function of the immersion time. An increase in the surface porosity as a function of immersion time was observed. For specimens with a longer immersion time (81 and 100 days) the surface changed and the roundly pores were replaced with an opening breach in the surface. This is an evidence of material degradation, since the difference between the control and the specimens increases with longer immersion times. The data obtained in the TGA analysis are presented in Fig. 4.
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Fig. 1. SEM micrographs of SEVA-C surfaces: (a) control and (b) after 13 immersion days (1500) (the images present dried samples after the immersion time).
Fig. 2. SEM micrographs of SEVA-C surfaces: (a) after 35 days and (b) after 48 immersion days (1500) (the images present dried samples after the immersion time).
The TGA results suggest that SEVA-C presents a water absorption capacity around 7% (w/w) until 60 days of immersion. After 2 months, the percent of water absorption increased to 10% and seems to stabilize thereafter. This phenomenon may be related with the degradation of the material, namely with the increase in the surface porosity with the immersion time. Fig. 5 shows the variation of contact angles with immersion time, using water as testing liquid. The contact angles results seem to tend toward constant values after the 120 days of immersion. As expected, the polar character of the surface did not change with the interaction with the solution.
The water uptake curves (Fig. 6) showed that SEVAC has a maximum of water absorption, stabilizing after the first 5 immersion days at 37 C. 3.2. Effect of immersion time on mechanical properties Figs. 7 and 8 presents the E1% and the strain at break of SEVA-C as a function of the immersion time (0, 3, 7, 14 and 30 days). The results evidenced a loss of the stiffness (Fig. 7) and, simultaneously, an increase in the ductility (Fig. 8) of the samples within the first days of immersion. These facts may be interpreted in terms of plasticity effect of
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Fig. 3. SEM micrographs of SEVA-C surfaces: (a) after 81 days and (b) after 100 immersion days (1500) (the images present dried samples after the immersion time).
Fig. 6. Water uptake of SEVA-C per initial specimen mass (1.13 g) in 50 ml of solution as function of immersion time. Fig. 4. SEVA-C water absorption versus immersion time (data obtained for TGA analysis).
Fig. 5. Contact angle measurements for water on SEVA-C, as a function of immersion time (measurements in dried samples).
Fig. 7. E-modulus of SEVA-C, as a function of immersion time in a sterile isotonic solution (NaCl 9 g/l).
the polymeric chains, resulting from the increment of the amount of up-taken water (Fig. 6), that acts as a plasticiser. It should be also stressed that although all the specimens were stabilised prior to mechanical testing, the amount of structural water within the starch phase can be higher in samples with longer immersion times. The results also show that after the first 3–4 days of immersion, the material performs a different degradation process characterised by a stabilisation of the stiffness (Fig. 7) and a decrease in the ductility (Fig. 8).
These effects should be associated with the release of almost all the plasticisers added to the material formulation and to the fact that the lubrication action of the absorbed water is no longer able to compensate the loss of these additives. 3.3. Evaluation of in-vitro degradation of SEVA-C The weight loss data during the immersion in a sterile isotonic solution of SEVA-C samples (Fig. 9) disclose two main stages.
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Fig. 8. Strain at break of SEVA-C, as a function of immersion time in a sterile isotonic solution (NaCl 9 g/l).
Fig. 10. Mass of glycerol released to the solution per initial specimen mass (1.61 g) in 50 ml of solution as function of immersion time.
Fig. 9. Weight loss of SEVA-C per initial specimen mass (1.13 g) in 50 ml of solution as function of immersion time.
Fig. 11. Mass of starch released to the solution per initial specimen mass (1.61 g) in 50 ml of solution as function of immersion time.
The first one, between 0 and 3–4 days, is related with combined physical phenomena including the leaching of plasticisers and other degradation products (such as low molecular weight polymeric chains) to the solution. After the 3rd day it is noticeable an unexpected decrease of the weight loss which was confirmed in several tests. The decrease in the ductility observed in Fig. 8 may allow to speculate that this weight loss variation could be associated to the establishment of hydrogen bonds between the adsorbed water and the polymeric chains of SEVA-C. For longer immersion times, the registered changes in the weight loss were less important, and can be related with the degradation of the polymeric matrix. In order to identify possible degradation products of the material, several complementary analysis were performed. The leaching of glycerol to the solution (Fig. 10) was the main physical phenomena. The leaching of glycerol to the solution increased in the first days, stabilizing thereafter, until the end of the assay. The mass of glycerol released to the solution per initial specimen mass is around 10%. This is the main component released to the solution, since the amount of released starch and polysaccharides was very low. It is also important to study the effects of the degradation solution on the chemical structure of the selected blends (Fig. 11). The percent of starch mass released to the solution as compared with the initial mass of the specimen is very
low (around 0.006%) and showed a small variation. After 2 months of immersion this quantity seems to increase a little, up to 0.008%. In order to quantify the total polysaccharides, the Dubois method was used. All the polysaccharides in solution were hydrolysed to monosaccharides, which after reaction with phenol, were able to be identified by spectrophotometry (Fig. 12). The percent mass of polysaccharides released to the solution, increased from the beginning, but this quantity is rather small, around 0.13%, until the end of the assay. The analytical errors associated with the starch and polysaccharide determinations were very small (between 0.57 and 1.37%). This is the reason why error bars were not plotted in the respective figures.
Fig. 12. Mass of total polysaccharides released to the solution measured by Dubois method [21] per initial specimen mass (1.61 g) in 50 ml of solution as function of immersion time.
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Fig. 13. Chromatogram of a SEVA-C sample extracted on Soxtec during 12 h.
In the first immersion days, the leaching of plasticisers, mainly glycerol, was the main physical degradation phenomenon. Monosaccharides and disaccharides, including maltodextrin, were not detected by HPAE-PAD along all the immersion time. This fact was expected since the starch phase cannot be degraded to small units. As the glycerol concentration in the degradation solutions was very high, the saccharides present in the solution could be ‘‘hindered’’ by the saturated peak of glycerol. No maltodextrin was detected in the chromatograms of the degradation solutions. As amylopectin is the only maltodextrin containing compound, this fact reinforces the idea that amylopectin is coming out at a slower rate than amylose. Anyway, it should be noted that, it is possible that with a living tissue with enzymatic activity, starch metabolic hydrolysis might occur. The FTIR experiments confirmed the increased presence of polysaccharides in the degradation solution, due to an increase of the ring frequency (1200–1000 cm 1) as a function of immersion time. 3.4. Total extraction — Soxtec apparatus The resulting solution from the Soxtec extraction was analysed in terms of glycerol concentration and carbohydrate content. The extraction time was approximately 12
h. The maximum extracted glycerol was approximately 13 3% mass of glycerol released/initial specimen mass. The previous results for glycerol indicated a leaching of approximately 10% mass of glycerol released/initial specimen mass) (Fig. 10). A joint analysis of both results suggest that a higher amount of glycerol in solution for longer immersion times should be expected. Simultaneously, the carbohydrate content was evaluated and the chromatograms allowed for the identification of several sugar derivatives (Fig. 13). The chromatograms were compared with numerous standards of glucopyranosyl derivatives. The retention time of 5.38 min corresponds to the elution of maltodextrin. The other peaks, which could be related with dextrin derivatives, were not possible to identify since high purity standards are not available (Fig. 13).
4. Conclusions Different stages can be identified in the in-vitro degradation behaviour of SEVA-C. The first one, between 0 and 3–4 days, is related with combined physical phenomena including the leaching of plasticisers and other degradation products to the solution, such as low molecular weight polymeric chains. After the 3rd day it is noticeable an unexpected decrease of the weight loss
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that was confirmed in several tests. For longer immersion times, the registered changes in the weight loss were less sensitive, and can be related with the degradation of the polymeric matrix. SEVA-C presents a water absorption capacity around 7% (w/w) until 60 days of immersion. After 2 months the percent of water absorption increased to 10%. The changes in SEVA-C internal structure can also be characterised by an increase in the surface porosity and ductility and a decrease in the stiffness. From the physical–chemical point of view the material degradation is mostly associated with the leaching of glycerol, the other metabolites were not released. Since the mechanical behaviour of SEVA-C is very sensitive to degradation, the understanding of the main relevant mechanisms and its control will allow for the production of a material with a mechanical performance appropriate for temporary biomedical applications.
Acknowledgements M.A. Arau´jo wishes to acknowledge Programme PRAXIS XXI for a research grant that supports her work. We are also grateful to Novamont, Italy, for supplying the blend used in this research and to Pronefro, Portugal, for performing the EtO sterilisations. References [1] Danniels AU, Andriano KP, Smutz WP, Chang MKO, Heller J. Evaluation of absorbable poly(ortho esters) for use in surgical implants. J Appl Biomater 1994;5:51. [2] Andriano KP, Pohojen T, Tomalla P. Processing and characterisation of absorbable polylactide polymers for use in surgical implants. J Appl Biomater 1994;5:133. [3] Baumgart FW, Perren SM. In: Harvey JP, Games RF, editors. Clinical and laboratory performance of bone plates. Philadelphia: STP1217. ASTM, 1994. p. 42. [4] Reis RL, Mendes SC, Cunha AM, Bevis MJ. Polymer and in vitro degradation of starch/EVOH thermoplastic blends. M Polym Int 1997;43:347.
[5] Reis RL, Cunha AM. Characterization of two biodegradable polymers of potential application within the biomaterials field. J Mater Sci: Mater Med 1995;6:786. [6] Reis RL, Cunha AM, Allan PS, Bevis MJ. Mechanical behavior of injection-molded starch-based polymers. Polym Adv Technol 1996;7:784. [7] Bastioli C, Bellotti V, Del Giudice L, Lombi R. PCT Int. Pat. Appl., W091/02025, 1991. [8] Bhattarcharya M, Vaidya UR, Zhang D, Narayan RJ. Properties of blends of starch and synthetic polymers containing anhydride groups. II. Effect of amilopectin to amylose ratio in starch. Appl Polym Sci 1995;57:539. [9] Bastioli C, Belotti V, Del Guidice L, Gilli G. Mater-bi: properties and biodegradability. J Environ Polym Degrad 1993;1:181. [10] Bastioli C, Bellotti V, Rallis A. Microstruture and melt flow behavior of a starch-based polymer. Rheol Acta 1994;33:307. [11] Griffin GJL. Starch polymer blends. Polym Degrad Stab 1994; 45:241. [12] Ratner BD. In: Ratner BD, Hoffman AS, editors. Biomaterials science: an introduction to materials in medicine, biomaterial science: an interdisciplinary endeavor. New York: Academic Press, 1996. [13] Ratner BD. In: Ratner BD, Hoffman AS, editors. Biomaterials science: an introduction to materials in medicine, biomedical applications of synthetic polymers. New York: Academic Press, 1996. p. 201. [14] Wool RP. In: Scott G, Gilead D, editors. The science and engineering of polymer composites in degradable polymers: principles and applications. London: Chapman and Hall, 1995. p. 138. [15] Williams DF. Mechanisms of biodegradation of implantable polymers. Clin Mater 1992;10:9. [16] Tjong SC, Bei JZ. Degradation behavior of poly(caprolactone)– poly(ethylene glycol) block copolymer/low-density polyethylene blends. Polym Eng Sci 1998;38:3. [17] Miller ND, Williams DF. On the degradation of PHB and PHB– PHV copolymers. Biomaterials 1987;8:129. [18] Zaikov GE. In: Jellinek HHG, editor. Degradation and stabilisation of polymers, vol. 2: biodegradation of polymers: kinetics and mechanisms. Amsterdam: Elsevier, 1989. p. 469. [19] Bastiolli C, Cerutti A, Guanella I, Romano GC, Tosin M. Physical state and biodegradation behavior of starch–polycaprolactone systems. J Environ Polym Degrad 1995;3:2. [20] Soest JJG, Benes K, Wit D, Vliegenthart JFG. The influence of starch molecular mass on the properties of extruded thermoplastic starch. Polymer 1996;37:3543. [21] Dubois M, Gilles KA, Hamilton JK, Rebens PA, Smith F. Colorimetric method for determination of sugars and related substances. Anal Chem 1956;28:350–5.
In-vitro degradation behaviour of starch/EVOH biomaterials M. Alberta Arau´jo a, Cla´udia M. Vaz b, Anto´nio M. Cunha b, Manuel Mota a,* a
Centro de Engenharia Biolo´gica (IBQF), Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal Department of Polymer Engineering, Universidade do Minho, Campus de Azure´m, 4800-058 Guimara˜es, Portugal
b
Received 28 June 2000; received in revised form 8 January 2001; accepted 17 January 2001
Abstract Blends of corn starch with poly(ethylene–vinyl alcohol) copolymer (SEVA-C) were aged up to 120 days on NaCl sterile isotonic solution (9 g/l) and the degradation solutions were analysed by several techniques. Mechanical properties (i.e. strength, stiffness and ductility), weight loss, and water uptake were also assessed. The evolution of the material structure was followed by scanning electron microscopy (SEM), contact angle measurements and thermogravimetric analysis (TGA). The degradation solutions were monitored with anionic chromatography (HPAE-PAD), colorimetric methods, and Fourier transformed infra-red spectrophotomety (FT-IR) in order to quantify the amount of plasticisers and sugars. SEVA-C micrographs presented an increase in the surface porosity as a function of immersion time. The main compound released to the solution is glycerol, the other degradation products are low molecular weight polymeric chains. From the physical–chemical point of view the material degradation is principally associated with the leaching of glycerol, since the other metabolites were not released. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Biodegradable polymer; In-vitro degradation; Biomedical; Starch/poly(ethylene–vinyl alcohol)
1. Introduction Bioabsorbable polymers may be advantageous for orthopaedic fracture fixation devices, mainly if they present an appropriate combination of initial strength and stiffness with a controlled degradation rate. The expectable reduction in the modulus of the biomaterial should lead to a gradual load transfer to the healing bone, and eventually, the complete absorption of the implant may avoid a second operation to its removal [1–3]. Systems based on poly(lactic acid) (PLA) [1,2], poly (glycolic acid) (PGA) [1] and their copolymers and poly(hydroxybutyrate) (PHB) [1,2] are well known examples of biodegradable polymers in orthopaedics. Blends of native corn starch with poly(ethylene–vinyl alcohol) copolymer (SEVA-C), are potential alternatives to the referred polymers used in clinical applications [4–6]. This class of materials has been reported as being biodegradable [7–11] but only recently were proposed for possible use as biomaterials [4–6]. The great advantage of this kind of materials as compared with the synthetic ones, is their * Corresponding author. Tel.: +351-253-604405; fax: +351-253678986. E-mail address: [email protected] (M. Mota).
biocompatibility with the organic tissues and their similarity with the bone dynamic mechanical behaviour. Certain performance requirements must be imposed upon materials intended to be used in contact with living systems [12]. Many of these specifications naturally fall into two categories: the effect of the organism on the implant and the effect of the implant on the organism [13]. These minimum standards include numerous criteria such as: (i) the material must not leach or release soluble components into the living systems, unless this release is intentional and/or non-toxic; (ii) the living system must not degrade the implant, unless this degradation is intentional [13]; (iii) the mechanical and physical behaviour of the polymer must be appropriate for the intended function and the desired mechanical properties must persist during the expected life of the implant; and finally (iv) the materials must be biocompatible, sterilisable and free of adherent bacteria and endotoxins from the bacteria cell walls [13]. In general, a polymeric material must be specifically engineered to meet these stringent demands [13] being the polymer degradability one of the most important factors leading to the acceptability of polymers for medical use [15]. Degradation, which can be defined as a damage on the chemical structure or a loss on physical
0141-3910/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0141-3910(01)00053-2
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properties or appearance of a plastic compound [14], can include [13]: polymeric chain cleavage (reduction in molecular weight), leaching of plasticisers and additives, chain crosslinking (increase in effective molecular weight), side chain hydrolysis or reaction and inappropriate swelling (from water or lipids). The in-vitro and in-vivo degradation of polymeric systems can be affected by various factors such as: polymer chemical structure and molecular mass, formulation and morphology, and thickness of the used specimen [16]. In most situations, degradation is undesirable since the device will be intended to remain in the body for a long period of time without any change in its functional properties and without inducing any significant adverse response from the tissues [15]. In other cases, however, degradation may be intentional, if either the function is transient so that the device requires elimination at an appropriate time, or if the function relies upon some degenerative process, as in drug or other biologically active agent release [17,18]. The degradation behaviour of insoluble polymers depends on both chemical structure and physical state, being of major importance the hydrophilicity (as in the case of starch based materials) [19,20]. Since the aqueous environment of the body remains reasonably constant (pH=7.4 and T=37 C) it should be possible to predict the susceptibility and kinetics of the in-vivo degradation process, considering hydrolysis the only mechanism present [15]. The in-vitro degradation tests, in physiological isotonic solutions, are able to simulate the interactions between the body fluids and a biomaterial, and can allow for the understanding of its stability and degradation rate. Despite being impossible to completely simulate the chemical, mechanical and dynamic character of the in-vivo system in order to predict the behaviour of biomaterials in the human physiological environment, in-vitro and in-vivo studies form an integral part of testing potential implant materials. Although biodegradable polymers are preferentially composed of residues normally present in the human body, implants made of these materials often trigger inflammatory responses. This paper presents a preliminary approach to the study of the mechanisms involved in the in-vitro degradation of a biomaterial based on starch/poly(ethylene–vinyl alcohol) blend, known as an alternative to the polymers currently used in clinical applications.
2. Materials and methods 2.1. Materials The material studied was a thermoplastic blend of corn starch with a poly(ethylene–vinyl alcohol) copolymer (60/40 mol/mol), SEVA-C, supplied by Novamont,
Novara, Italy. Two different types of specimens (tensile bars and square plates, with cross sections of 24 and 230 mm, respectively) were moulded in a Klockner Ferromatic FM-20 injection machine. The SEVA-C samples, were weighed and immersed for several pre-fixed ageing periods (until 120 days) in a sterile isotonic solution (NaCl 9 g/l) at 37 C and pH=7.4. The tensile bars were used to evaluate the material water uptake and weight loss and also for mechanical testing. The plates were used in in-vitro degradation experiments under strictly controlled conditions in a laminar flux chamber. The samples were sterilized by autoclave in an atmosphere of 12/88 mixture of ethylene oxide (EtO) and carbon dioxide (CO2), with a cycle time of 14 h at a working temperature of 45 C, a moisture level of 50% and a chamber pressure of 50 kPa. 2.2. Physical and mechanical characterization After being removed from the degradation solutions (50 ml), the tensile bars were immediately weighed to evaluate the respective water uptake. One batch (three specimens of about 1.300 g each) was dried up to exhaustion in an oven for 72 h at 70 2 C in order to determine the respective weight loss. Another batch was conditioned in a chamber with a controlled atmosphere (23 C and 55% RH) for 1 week, to stabilise the moisture content. In order to evaluate the changes in the mechanical properties as a function of the immersion time, the specimens from the conditioned batch were tested in an Instron 4505 universal machine, fitted with a resistive extensometer (gauge length 10 mm), in a controlled environment (23 C and 55% RH). The crosshead velocity was 8.310 5 m/s (5 mm/min) until 1% strain and then increased to 8.310 4 m/s (50 mm/min) until fracture. These tests were aimed at determining the ultimate tensile strength (UTS), the secant modulus at 1% strain (E1%) and the strain at break ("r). 2.3. Analytical and morphologycal methods 2.3.1. Thermogravimetric analysis Thermogravimetric analysis (TGA) was performed in a TGA-50 Shimadzu-thermogravimetric equipment using aluminium pans, under the following conditions: heating rate of 5 C/min, hold temperature and time of 120 C and 110 min, respectively, and a constant helium flow of 30 ml/min. Samples of about 18 mg were used for the TGA tests. The samples were removed for testing at pre-defined immersion time (until 120 days) and kept in a exsicator, with controlled temperature and humidity, until constant weight. All the assays were performed in triplicate, considering the medium of the three results as the final result.
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2.3.2. Detection of starch and polysaccharides The starch amount in the degradation solutions was determined by means of a simple colorimetric method. 300 ml of sulphuric acid 2 M and 0.5 ml of KI-I2 were added to a 5 ml of sample of the degradation solution. The absorbance of the resulting solution was determined at a wavelength of 580 nm, being the respective concentration obtained from a standard curve, obtained with the same corn starch used in the samples. The total amount of polysaccharides in the degradation solutions was quantified using the Dubois method [21] which is based on the addition of 1 ml of phenol (5% w/v) and 5 ml sulphuric acid (95–97%) to 1 ml of sample of the degradation solution. The absorbance of the resulting mixture was determined at a wavelength of 490 nm using water in the reference cell. 2.3.3. Detection of saccharides and glycerol with high performance anion exchange chromatography High performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD) equipped with a Carbopac PA1 (4250 mm) column and a Carbopac guard column (450 mm) was used to separate saccharides and glycerol from the degradation solutions. Separation was performed with a flow rate of 1 ml/ min using an eluent gradient of 50% of NaOH 0.2 M and ultra-pure water (referred as the 50% gradient). Eluents were flushed with helium to remove dissolved gases and were continuously pressurised with the eluent degas module of Dionex. Another gradient of 20% NaOH and 80% water was also used to elute the monosaccharides and disaccharides after the Soxtec extraction described below. This gradient was applied to separate glycerol and saccharides from all the degradation solutions at different times. Lactose (50 mg/l) was used as the internal standard. 2.3.4. Soxtec extraction Total extraction was used to isolate soluble matter, such as additives, from the studied material. The extraction was performed in a Soxtec system HT 2 Tecator during 12 h. SEVA-C square specimens were weighed into thimbles and inserted into the extraction unit. After solvent addition (water was used as the extraction medium) to the extraction cups, the soluble material was extracted in a two stage process. First, the sample was immersed in boiling water to dissolve most of the soluble material. In the second step, the sample was raised above the solution to enable an efficient washing with water from the condensers. Finally, the extraction cups were dried and weighed. The obtained extracted solutions were injected in HPAE-PAD using the 50% gradient described above to analyse the glycerol and saccharide content.
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2.3.5. FTIR Degradation solutions, of different ageing periods, were freeze dried in a Freeze Dryer Alpha 2-4. The drying was achieved by avoiding the liquid state through sublimation using the following procedure: (i) pre-freezing the degradation solutions; (ii) freeze-drying (24 h) according to a pre-selected temperature ( 70 C) and vacuum level (0.05 mbar); and (iii) final drying of products to remove the capillary and molecular bound water. After freeze drying, the resulting powders were conditioned in an exsicator before the preparation in KBr pellets for FTIR analysis. FTIR assays were performed in a Perkin-Elmer FT-IR 1600 spectrophotometer. 2.3.6. Contact angle measurements The plates were dried at 80 C for 1 week and kept in a exsicator. Contact angle measurements were carried out at room temperature using the sessile drop technique, with a video camera mounted on a microscope to record the drop image. Water was used for this purpose. Sessile drops were deposited with a micrometric syringe directly on the surface from that metallic needle. Pictures of the drops were taken, at regular intervals in a standard contact angle apparatus. At least 30 drops of about 6 ml for each sample were analysed. 2.3.7. SEM The effect of the solution on the surface morphology of the SEVA-C specimens, as a function of immersion time, was followed by scanning electron microscopy (SEM) in a Leica Cambridge S360 microscope. The plates were dried at 80 C for 1 week and kept in a exsicator before the analysis. For control, a non-immersed specimen, stored at controlled environment conditions was used.
3. Results 3.1. Thermogravimetric behaviour and surface morphology of SEVA-C The change of the material surface with the immersion time was morphologically assessed by SEM. The SEVA-C surfaces micrographs (Figs. 1–3) showed the change of the material surface as a function of the immersion time. An increase in the surface porosity as a function of immersion time was observed. For specimens with a longer immersion time (81 and 100 days) the surface changed and the roundly pores were replaced with an opening breach in the surface. This is an evidence of material degradation, since the difference between the control and the specimens increases with longer immersion times. The data obtained in the TGA analysis are presented in Fig. 4.
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Fig. 1. SEM micrographs of SEVA-C surfaces: (a) control and (b) after 13 immersion days (1500) (the images present dried samples after the immersion time).
Fig. 2. SEM micrographs of SEVA-C surfaces: (a) after 35 days and (b) after 48 immersion days (1500) (the images present dried samples after the immersion time).
The TGA results suggest that SEVA-C presents a water absorption capacity around 7% (w/w) until 60 days of immersion. After 2 months, the percent of water absorption increased to 10% and seems to stabilize thereafter. This phenomenon may be related with the degradation of the material, namely with the increase in the surface porosity with the immersion time. Fig. 5 shows the variation of contact angles with immersion time, using water as testing liquid. The contact angles results seem to tend toward constant values after the 120 days of immersion. As expected, the polar character of the surface did not change with the interaction with the solution.
The water uptake curves (Fig. 6) showed that SEVAC has a maximum of water absorption, stabilizing after the first 5 immersion days at 37 C. 3.2. Effect of immersion time on mechanical properties Figs. 7 and 8 presents the E1% and the strain at break of SEVA-C as a function of the immersion time (0, 3, 7, 14 and 30 days). The results evidenced a loss of the stiffness (Fig. 7) and, simultaneously, an increase in the ductility (Fig. 8) of the samples within the first days of immersion. These facts may be interpreted in terms of plasticity effect of
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Fig. 3. SEM micrographs of SEVA-C surfaces: (a) after 81 days and (b) after 100 immersion days (1500) (the images present dried samples after the immersion time).
Fig. 6. Water uptake of SEVA-C per initial specimen mass (1.13 g) in 50 ml of solution as function of immersion time. Fig. 4. SEVA-C water absorption versus immersion time (data obtained for TGA analysis).
Fig. 5. Contact angle measurements for water on SEVA-C, as a function of immersion time (measurements in dried samples).
Fig. 7. E-modulus of SEVA-C, as a function of immersion time in a sterile isotonic solution (NaCl 9 g/l).
the polymeric chains, resulting from the increment of the amount of up-taken water (Fig. 6), that acts as a plasticiser. It should be also stressed that although all the specimens were stabilised prior to mechanical testing, the amount of structural water within the starch phase can be higher in samples with longer immersion times. The results also show that after the first 3–4 days of immersion, the material performs a different degradation process characterised by a stabilisation of the stiffness (Fig. 7) and a decrease in the ductility (Fig. 8).
These effects should be associated with the release of almost all the plasticisers added to the material formulation and to the fact that the lubrication action of the absorbed water is no longer able to compensate the loss of these additives. 3.3. Evaluation of in-vitro degradation of SEVA-C The weight loss data during the immersion in a sterile isotonic solution of SEVA-C samples (Fig. 9) disclose two main stages.
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Fig. 8. Strain at break of SEVA-C, as a function of immersion time in a sterile isotonic solution (NaCl 9 g/l).
Fig. 10. Mass of glycerol released to the solution per initial specimen mass (1.61 g) in 50 ml of solution as function of immersion time.
Fig. 9. Weight loss of SEVA-C per initial specimen mass (1.13 g) in 50 ml of solution as function of immersion time.
Fig. 11. Mass of starch released to the solution per initial specimen mass (1.61 g) in 50 ml of solution as function of immersion time.
The first one, between 0 and 3–4 days, is related with combined physical phenomena including the leaching of plasticisers and other degradation products (such as low molecular weight polymeric chains) to the solution. After the 3rd day it is noticeable an unexpected decrease of the weight loss which was confirmed in several tests. The decrease in the ductility observed in Fig. 8 may allow to speculate that this weight loss variation could be associated to the establishment of hydrogen bonds between the adsorbed water and the polymeric chains of SEVA-C. For longer immersion times, the registered changes in the weight loss were less important, and can be related with the degradation of the polymeric matrix. In order to identify possible degradation products of the material, several complementary analysis were performed. The leaching of glycerol to the solution (Fig. 10) was the main physical phenomena. The leaching of glycerol to the solution increased in the first days, stabilizing thereafter, until the end of the assay. The mass of glycerol released to the solution per initial specimen mass is around 10%. This is the main component released to the solution, since the amount of released starch and polysaccharides was very low. It is also important to study the effects of the degradation solution on the chemical structure of the selected blends (Fig. 11). The percent of starch mass released to the solution as compared with the initial mass of the specimen is very
low (around 0.006%) and showed a small variation. After 2 months of immersion this quantity seems to increase a little, up to 0.008%. In order to quantify the total polysaccharides, the Dubois method was used. All the polysaccharides in solution were hydrolysed to monosaccharides, which after reaction with phenol, were able to be identified by spectrophotometry (Fig. 12). The percent mass of polysaccharides released to the solution, increased from the beginning, but this quantity is rather small, around 0.13%, until the end of the assay. The analytical errors associated with the starch and polysaccharide determinations were very small (between 0.57 and 1.37%). This is the reason why error bars were not plotted in the respective figures.
Fig. 12. Mass of total polysaccharides released to the solution measured by Dubois method [21] per initial specimen mass (1.61 g) in 50 ml of solution as function of immersion time.
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Fig. 13. Chromatogram of a SEVA-C sample extracted on Soxtec during 12 h.
In the first immersion days, the leaching of plasticisers, mainly glycerol, was the main physical degradation phenomenon. Monosaccharides and disaccharides, including maltodextrin, were not detected by HPAE-PAD along all the immersion time. This fact was expected since the starch phase cannot be degraded to small units. As the glycerol concentration in the degradation solutions was very high, the saccharides present in the solution could be ‘‘hindered’’ by the saturated peak of glycerol. No maltodextrin was detected in the chromatograms of the degradation solutions. As amylopectin is the only maltodextrin containing compound, this fact reinforces the idea that amylopectin is coming out at a slower rate than amylose. Anyway, it should be noted that, it is possible that with a living tissue with enzymatic activity, starch metabolic hydrolysis might occur. The FTIR experiments confirmed the increased presence of polysaccharides in the degradation solution, due to an increase of the ring frequency (1200–1000 cm 1) as a function of immersion time. 3.4. Total extraction — Soxtec apparatus The resulting solution from the Soxtec extraction was analysed in terms of glycerol concentration and carbohydrate content. The extraction time was approximately 12
h. The maximum extracted glycerol was approximately 13 3% mass of glycerol released/initial specimen mass. The previous results for glycerol indicated a leaching of approximately 10% mass of glycerol released/initial specimen mass) (Fig. 10). A joint analysis of both results suggest that a higher amount of glycerol in solution for longer immersion times should be expected. Simultaneously, the carbohydrate content was evaluated and the chromatograms allowed for the identification of several sugar derivatives (Fig. 13). The chromatograms were compared with numerous standards of glucopyranosyl derivatives. The retention time of 5.38 min corresponds to the elution of maltodextrin. The other peaks, which could be related with dextrin derivatives, were not possible to identify since high purity standards are not available (Fig. 13).
4. Conclusions Different stages can be identified in the in-vitro degradation behaviour of SEVA-C. The first one, between 0 and 3–4 days, is related with combined physical phenomena including the leaching of plasticisers and other degradation products to the solution, such as low molecular weight polymeric chains. After the 3rd day it is noticeable an unexpected decrease of the weight loss
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that was confirmed in several tests. For longer immersion times, the registered changes in the weight loss were less sensitive, and can be related with the degradation of the polymeric matrix. SEVA-C presents a water absorption capacity around 7% (w/w) until 60 days of immersion. After 2 months the percent of water absorption increased to 10%. The changes in SEVA-C internal structure can also be characterised by an increase in the surface porosity and ductility and a decrease in the stiffness. From the physical–chemical point of view the material degradation is mostly associated with the leaching of glycerol, the other metabolites were not released. Since the mechanical behaviour of SEVA-C is very sensitive to degradation, the understanding of the main relevant mechanisms and its control will allow for the production of a material with a mechanical performance appropriate for temporary biomedical applications.
Acknowledgements M.A. Arau´jo wishes to acknowledge Programme PRAXIS XXI for a research grant that supports her work. We are also grateful to Novamont, Italy, for supplying the blend used in this research and to Pronefro, Portugal, for performing the EtO sterilisations. References [1] Danniels AU, Andriano KP, Smutz WP, Chang MKO, Heller J. Evaluation of absorbable poly(ortho esters) for use in surgical implants. J Appl Biomater 1994;5:51. [2] Andriano KP, Pohojen T, Tomalla P. Processing and characterisation of absorbable polylactide polymers for use in surgical implants. J Appl Biomater 1994;5:133. [3] Baumgart FW, Perren SM. In: Harvey JP, Games RF, editors. Clinical and laboratory performance of bone plates. Philadelphia: STP1217. ASTM, 1994. p. 42. [4] Reis RL, Mendes SC, Cunha AM, Bevis MJ. Polymer and in vitro degradation of starch/EVOH thermoplastic blends. M Polym Int 1997;43:347.
[5] Reis RL, Cunha AM. Characterization of two biodegradable polymers of potential application within the biomaterials field. J Mater Sci: Mater Med 1995;6:786. [6] Reis RL, Cunha AM, Allan PS, Bevis MJ. Mechanical behavior of injection-molded starch-based polymers. Polym Adv Technol 1996;7:784. [7] Bastioli C, Bellotti V, Del Giudice L, Lombi R. PCT Int. Pat. Appl., W091/02025, 1991. [8] Bhattarcharya M, Vaidya UR, Zhang D, Narayan RJ. Properties of blends of starch and synthetic polymers containing anhydride groups. II. Effect of amilopectin to amylose ratio in starch. Appl Polym Sci 1995;57:539. [9] Bastioli C, Belotti V, Del Guidice L, Gilli G. Mater-bi: properties and biodegradability. J Environ Polym Degrad 1993;1:181. [10] Bastioli C, Bellotti V, Rallis A. Microstruture and melt flow behavior of a starch-based polymer. Rheol Acta 1994;33:307. [11] Griffin GJL. Starch polymer blends. Polym Degrad Stab 1994; 45:241. [12] Ratner BD. In: Ratner BD, Hoffman AS, editors. Biomaterials science: an introduction to materials in medicine, biomaterial science: an interdisciplinary endeavor. New York: Academic Press, 1996. [13] Ratner BD. In: Ratner BD, Hoffman AS, editors. Biomaterials science: an introduction to materials in medicine, biomedical applications of synthetic polymers. New York: Academic Press, 1996. p. 201. [14] Wool RP. In: Scott G, Gilead D, editors. The science and engineering of polymer composites in degradable polymers: principles and applications. London: Chapman and Hall, 1995. p. 138. [15] Williams DF. Mechanisms of biodegradation of implantable polymers. Clin Mater 1992;10:9. [16] Tjong SC, Bei JZ. Degradation behavior of poly(caprolactone)– poly(ethylene glycol) block copolymer/low-density polyethylene blends. Polym Eng Sci 1998;38:3. [17] Miller ND, Williams DF. On the degradation of PHB and PHB– PHV copolymers. Biomaterials 1987;8:129. [18] Zaikov GE. In: Jellinek HHG, editor. Degradation and stabilisation of polymers, vol. 2: biodegradation of polymers: kinetics and mechanisms. Amsterdam: Elsevier, 1989. p. 469. [19] Bastiolli C, Cerutti A, Guanella I, Romano GC, Tosin M. Physical state and biodegradation behavior of starch–polycaprolactone systems. J Environ Polym Degrad 1995;3:2. [20] Soest JJG, Benes K, Wit D, Vliegenthart JFG. The influence of starch molecular mass on the properties of extruded thermoplastic starch. Polymer 1996;37:3543. [21] Dubois M, Gilles KA, Hamilton JK, Rebens PA, Smith F. Colorimetric method for determination of sugars and related substances. Anal Chem 1956;28:350–5.