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In vitro biocompatibility of biodegradable dextran-based hydrogels tested with human "broblasts

Cees J. De Groot *, Marja J.A. Van Luyn , Welmoed N.E. Van Dijk-Wolthuis , Jenny A. Cade H e , Jose H e A. Plantinga , Willem Den Otter , Wim E. Hennink
Department of Cell Biology and Histology, Faculty of Veterinary Medicine, Utrecht University, P.O. Box 80 176, 3508 TD Utrecht, Netherlands Department of Pathology Lab Medicine, Medical Biology, Cell Biology and Biomaterials, Faculty of Medical Sciences, University of Groningen, Bloemsingel 10, 9712 KZ Groningen, Netherlands Department of Pharmaceutics, Faculty of Pharmacy, Utrecht University, P.O. Box 80 082, 3508 TB Utrecht, Netherlands Received 4 February 2000; accepted 3 August 2000

Abstract The cytotoxicity of dextran T40, methacrylated dextran (dex-MA) and hydroxyethyl-methacrylated dextran (dex-HEMA), dextran-based hydrogel discs and microspheres, and their degradation products, was studied by measuring the cell proliferation inhibition index (CPII) on human "broblasts in vitro. In addition, during the 72 h incubation period light-microscopic observations were performed daily. After 24 h of incubation with dextran and dex-HEMA polymers, the cells showed elongated or spider-like forms, some lipid droplets and intracellular granula, indicative of pinocytosis and internalization of the polymers. During the next two days, the "broblasts' appearance did not change. Methacrylic acid (MAA), formed by hydrolysis of dex-HEMA, did not in#uence the cell morphology. Dex-HEMA polymer solutions with a low and high degree of substitution (DS) at 100 mg/ml caused a CPII of 30}40% after 72 h. This is less than 10% growth inhibition per cell cycle and statistically not di!erent from the CPII induced by 100 mg/ml dextran T40. Growth inhibition induced by MAA was also low. The various dex-MA hydrogel discs caused similar low growth inhibition. Interestingly, hydrogel microspheres of dex-MA and dex-(lactate-)HEMA caused a CPII of only 0}20% after 72 h. The results presented in this study demonstrate that methacrylate-derivatized dextran hydrogels show good biocompatibility in vitro making these degradable biomaterials promising systems for drug delivery purposes. 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Methacrylated dextrans; Biodegradable hydrogel; Biocompatibility; Fibroblasts

1. Introduction Recent advances in biotechnology have resulted in large-scale production of pharmaceutically active peptides and proteins, such as, for instance, insulin, growth hormones and cytokines [1]. E!ective application of protein drugs, however, requires special delivery methods [1]: simple oral administration is not possible because the protein drugs are rapidly degraded by the proteolytic enzymes and in the acidic environment of the gastrointestinal tract [2]. Furthermore, proteins have a short half-life in the blood circulation, which necessitates repeated injection or continuous infusion of the protein drug to obtain a pharmacological e!ect.
* Corresponding author. Tel.: #31-30-253-4338; fax: #31-30-2535862. E-mail address: c.j.degroot@vet.uu.nl (C.J. De Groot).

The therapeutic applicability of these pharmaceutically active proteins can be greatly increased by the use of controllable drug delivery systems like hydrogels. Hydrogels are three-dimensional networks of hydrophilic polymers, which can absorb substantial amounts of water. In general, hydrogels possess a good biocompatibility [3]. Their hydrophilic surface has a low interfacial free energy in contact with body #uids [4], which results in a low tendency of proteins and cells to adhere to these surfaces. Moreover, the soft and rubbery nature of hydrogels minimizes irritation of the surrounding tissue [5]. Hydrophobic domains on the surface of hydrogel materials can promote cell adhesion [6]. We have recently reported on the synthesis and characterization of methacrylate-derivatized dextran hydrogels for the controlled release of proteins: the non-biodegradable methacrylated dextran (dex-MA) hydrogels, and biodegradable (lactate)-hydroxyethyl

0142-9612/01/$ - see front matter 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 2 - 9 6 1 2 ( 0 0 ) 0 0 2 6 6 - 0

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methacrylated dextran (dex-(lactate-)HEMA) hydrogels [7,8]. Dex-MA hydrogels are stable under physiological conditions, whereas hydrogels derived from dex-(lactate)HEMA degrade at pH 7.2 and 373C, due to chemical hydrolysis of the lactate and carbonate ester bonds present in the crosslinks. The swelling and degradation rate of these hydrogels is dependent on the crosslink density, which can be varied by changing the degree of substitution (DS, the number of methacryloyl groups per 100 glucose units of the polymer) and/or the initial water content of the hydrogel [7}9]. The hydrogels are prepared in an all-aqueous system, thereby minimizing damage to protein drugs to be encapsulated. Furthermore, the protein release rate * based on the di!usion rate of the protein and/or the degradation rate of the hydrogel * can be controlled by varying the crosslink density of the hydrogels [7}9], enabling also the controlled release of low molecular weight proteins like cytokines. Since these hydrogels are intended to be used for delivery of pharmaceutically active peptides and proteins in vivo, it is necessary to have insight into the biocompatibility of these materials, both in vitro and in vivo. It is well known that the low molecular weight fractions of dextran ((110 kDa) are relatively inert and non-toxic. This allows their use as plasma expander [10]. Furthermore, dextran is widely under investigation as a polymeric carrier in novel drug delivery systems, both as macromolecular prodrug [11] and in hydrogels [12,13]. Polymethacrylates are used in a number of pharmaceutical and biomedical applications [14,15]. Although residual monomers are generally toxic, the polymers itself are usually well tolerated [15}17]. Furthermore, it was shown that copolymers of poly-HEMA and acrylated poly(ethylene glycol) were biocompatible as surface coating of a biosensor [18]. Recently, we have reported on the in vivo biocompatibility of dex-MA and dex-lactate-HEMA hydrogel discs [19]. In these studies the tissue reactions up to six weeks after subcutaneous implantation in rats of hydrogel discs with di!erent crosslink density have been investigated [19]. We now report on the results obtained with a sensitive in vitro cytotoxicity test of methacrylated dextrans, of degradation products, and of hydrogel discs and microspheres. These in vitro biocompatibility tests are required to evaluate the biological safety of new biomaterials [20]. According to the ISO norms [20], biomaterials should be evaluated at three levels: (1) the biological safety of the various ingredients used to manufacture the basic materials, (2) the biological safety of potential leachable substances or degradation products, and (3) the biological safety of the "nal biomaterial. The in vitro cytotoxicity was assessed with human skin "broblasts from an established cell line after 72 h of incubation. Moreover, the cellular morphology was monitored daily by light-microscopic examination.

2. Materials and methods 2.1. Materials Methacrylic acid (98.5#%, Acros Chimica, Geel, Belgium) was distilled under vacuum and stored at ! 303C before use. Dex-MA and dex-(lactate-)HEMA polymers with di!erent DS were synthesized as described [21}23]. Dextran (T40, from Leuconostoc mesenteroides), potassium peroxydisulfate (KPS) and N,N,N,N-tetramethylethylenediamine (TEMED) were obtained from Fluka Chemie AG, Buchs, Switzerland. Poly(ethylene glycol) (PEG; MW 10,000 g/mol) was obtained from Merck (Darmstadt, Germany). All other chemicals used were of analytical grade. Water was puri"ed by reversed osmosis. Phosphate-bu!ered saline (PBS; 10 mM, pH 7.2) used for the gel preparation, was deoxygenized before use by bubbling nitrogen gas through it. Dulbecco's modi"ed Eagle medium (DMEM) (Life Technologies, Breda, The Netherlands) was supplemented with 10% fetal bovine serum (Greiner, Alphen aan den Rijn, The Netherlands), 2 mM glutamine (Life Technologies), penicillin, and streptomycin, both 100 units/ml (Sigma Chemical Co., St. Louis, MO, USA). This medium is further referred to as DMEM complete medium. 2.2. Methods 2.2.1. Degradation of dex-MA in DMEM complete medium A solution of 10 mg/ml dex-MA DS 30 in DMEM complete medium pH 7.4 (including 25 mM HEPES (4-(2hydroxyethyl)-1-piperazine ethanesulfonic acid)) was sterilized by "ltration over a 0.22 m "lter and left at 373C. After 72 h, a 500 l sample was mixed with 500 l 6% w/v trichloroacetic acid in water. The precipitated proteins were removed by centrifugation, and the amount of methacrylic acid in the supernatant was quanti"ed with HPLC as previously described [24]. 2.2.2. Positive and negative control materials tested The control cultures only contained DMEM complete medium. Poly(urethane) "lm (thickness about 1 mm; made from 2363}55D-pellethane resin (Dow Chemical, Midland, MI, USA) by Medtronic Promeon, Minneapolis, MN, USA) was used as a negative control, and latex rubber (thickness 3}4 mm; Hilversum Rubber Factory, Hilversum, The Netherlands) as a positive control. From both materials discs with a diameter of 8 mm were cut, and routinely sterilized with ethylene oxide. 2.2.3. Preparation of hydrogel discs and microspheres Methacrylated dextran (150 mg, corresponding to a "nal concentration of 10% w/w) was dissolved in 1140 l PBS. To this solution 135 l of KPS in PBS (50 mg/ml; 17 mol KPS/g of gel) was added and mixed well. Subsequently, TEMED (75 l; 20% (v/v) in PBS, pH

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adjusted to 7 with 4 N HCl; 67 mol/g of gel) was added and the resulting solution was quickly transferred into the circular holes (diameter 10 mm, depth 2 mm) of a Delrin plate. The hydrogel discs were allowed to polymerize for 1 h at room temperature. For gels with 70% initial water content, 450 mg of methacrylated dextran was dissolved in 840 l PBS, after which the same procedure was followed. After polymerization, the slab-formed hydrogels were removed from the template, and subsequently equilibrated and extracted with water at 43C for 48 h. The hydrogel discs were placed in vials containing 30 ml of water (which was "ltered through a 0.22 m "lter), and steam sterilized for 30 min at 1213C. Subsequently, the discs were washed three times with sterile PBS and DMEM complete medium before application to the cell culture. Two discs were used per well, except for the highly swollen dex-MA DS 5 hydrogel with 90% initial water content, of which one disc per well was used. Microspheres with initial water content of 50 and 70%, using dex-MA with DS 5, 9 or 14 (PEG/dextran ratio of 7.5), polydisperse dex-lactate-HEMA DS 4.4 (PEG/dexratio of 10), and dex-HEMA DS 16 (PEG/dex-ratio of 5), were prepared by the two-phase aqueous emulsion technique as described previously [25,26]. Size distributions of microsphere preparations were determined with an Accusizer2+ (Model 770, Particle Sizing Systems, Santa Barbara, CA) and C770 software version 2.54. Volume mean particle diameters D[3,0], according to Edmundson [27], varied from 9.2 to 14.9 m. After washing three times with 100 mM phosphate bu!er pH 5 (i.e. no hydrolysis), the microspheres were autoclaved (15 min, 1213C) in the same bu!er. Before application as a 10% (wet weight) suspension, the microsphere preparations were washed twice with PBS pH 7.4. 2.2.4. Solutions of dextran, dex-MA, dex-HEMA, and MAA Solutions of 100 mg (hydroxyethyl)methacrylated dextran per ml, and 105 and 470 g MAA (bu!ered with 25 mM HEPES pH 7.4) per ml DMEM complete medium were made on the day of application, and sterilized by subsequent "ltration through a 0.45 and 0.22 m "lter. The osmolality of the solutions was measured using an Osmomat 030 (Gonotec, Berlin, Germany) osmometer. 2.2.5. Cultures Human skin "broblasts from the established cell line PK84 were routinely cultured in DMEM complete medium. The cells were incubated at 373C in air containing 5% CO . After washing twice with sterile PBS, human  "broblasts were harvested using 0.05% trypsin in Ca>/Mg>-free Hanks' balanced salt solution (HBSS; Sigma). Subsequently, the cells were centrifuged and resuspended in fresh DMEM complete medium. For all cultures, a "nal volume of 2 ml medium, containing 5;10 cells, was pipetted into each well of a six-well tissue-culture plate (Costar, Badhoevedorp, The Nether-

lands). Since each well has a surface area of 10 cm, the "nal seeding density was 5;10 cells/cm. All tests were set up in triplicate. After cell attachment (about 4 h after cell seeding) the tests were started in the following way. For testing dextran, dex-MA, dex-HEMA and MAA, the culture media were removed and the test solutions (2 ml) were added to the cells. For the control culture, the culture medium was refreshed, whereas for the tests of the hydrogel discs, the medium was refreshed and the discs were carefully placed in the wells. For testing microspheres, a 10% suspension (wet weight) of non-sized microspheres in complete medium was added. 2.2.6. Microscopy At days 1}3 cell growth and morphology were evaluated by inspection with phase-contrast inverted light microscopy. Cytotoxic e!ects were scored with respect to the degree of cellular lysis (i.e. number of #oating cells), impaired cell adhesion, induction of cellular granula, formation of lipid droplets, and other possible morphological changes. 2.2.7. Cell counts Seventy-two hours after starting the cultures, the discs or microspheres and the supernatants including detached cells were removed. The cell layer adhering to the bottom of each well was washed with PBS to remove remaining materials and loose cells. Subsequently, the cells were trypsinized, resuspended and counted using a Casy 1 model DT Counter (Schaerfe System GmbH, Reutlingen, Germany). The mean cell number from the three wells per tested material was calculated and used to determine the cell proliferation inhibition index (CPII), expressed as a percentage of the mean cell number in control cultures, using the following equation: CPII"100%!(mean cell number of test culture/ mean cell number of control culture;100%). Whether the calculated CPII for one material was signi"cantly di!erent from a value for another material was tested with the Student's t-test.

3. Results and discussion To evaluate the cytotoxicity of dextran, methacrylatederivatized dextrans, and dextran-based hydrogel discs and microspheres, as well as of their degradation products, we have determined the proliferation, morphological deviancies or cell death of human "broblasts incubated with these materials for three days. 3.1. Control cultures In the presence of poly(urethane), which served as the negative control, the cell growth was comparable to the

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Fig. 2. The cell proliferation inhibition index (mean%$SD) after 72 h for the polymers dextran, methacrylated dextran (dex-MA), and hydroxyethyl methacrylated dextran (dex-HEMA) with di!erent DS, and for the degradation product methacrylic acid (MAA). Data are from a representative experiment, performed in triplicate.

Fig. 1. Human "broblasts, cultured for (A) 24 h and (B) 72 h in the presence of poly(urethane) (negative control). After 24 h, cluster formation of cells is visible, whereas after 72 h an almost con#uent cell layer has formed (magni"cation 200;).

medium control cultures. At day 1, seeded cells were adhering and started to form clusters (Fig. 1A). After 72 h, an almost con#uent layer of cells was formed, and no morphological changes were observed (Fig. 1B). No cytotoxicity was found after incubation with polyurethane "lms (Fig. 2). In contrast, in the presence of latex rubber, which was used as a positive control, cell detachment and extensive cellular lysis occurred within the "rst 24 h (Fig. 3). Cell proliferation was not observed (Fig. 2; CPII'95%) and adhering cells were hardly found. 3.2. Solutions of dextran, dex-MA, dex-HEMA, and MAA Dextran T40 and dex-MA (DS 5 and 30) polymer solutions were incubated with "broblast cultures for 72 h at a concentration of 100 mg/ml, corresponding to the solid material of a hydrogel with an initial water content of 90%. Dialysed dextran exhibited a relative inhibition

Fig. 3. Human "broblasts, cultured for 24 h in the presence of latex rubber (positive control). The cells are dead and #oating in the medium (magni"cation 200;).

of 25$7% (Fig. 2). This is less than 10% growth inhibition per cell cycle, supposing a cell cycling time of approximately 24 h. In other studies, macrophages and bovine brain microvessel-related endothelial cells were incubated in vitro with dextran T70 for 24 h in a concentration of 0.1 and 1 mg/ml, respectively [28]. As measured by a lactate dehydrogenase (LDH) leakage assay and by microscopic observations, this biopolymer was found to be essentially non-toxic, i.e. dextran T70 induced approx. 10% cytotoxicity (LDH release) after 24 h incubation [28]. The CPII values of dex-MA DS 5 (41$13%; Fig. 2) and DS 30 (35$8%; Fig. 2) were in the 25}40% range after 72 h; this is a low cytotoxicity, not statistically signi"cantly di!erent from the CPII of dextran. Fig. 4 shows a photograph of cells after incubation with a

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Fig. 4. Human "broblasts, cultured for 24 h in the presence of dex-MA DS 5 (also representative for dextran and dex-MA DS 30). The cells have spider-like structures and contain intracellular granula (see arrows) (magni"cation 200;).

dex-MA DS 5 solution for 24 h, which is similar to the appearance of the cells after incubation with dextran and dex-MA DS 30. The cells showed elongated or spider-like forms, and contained some lipid droplets and intracellular granula. Using phase-contrast inverted light microscopy we can discriminate between the yellow}green appearing lipid droplets and the dark granula [29]. Furthermore, our LM observations have been extensively validated by studies applying transmission electron microscopy (TEM) [29]. The granula might be due to pinocytosis or phagocytosis of the tested materials, and/or of dead cells, whereas the presence of lipid droplets might indicate a slight fatty degeneration of the cells. In the in vivo biocompatibility studies [19] we have shown that polymers, pieces of hydrogel discs and small microspheres can be phagocytosed, as demonstrated by the speci"c staining of dextrans with the periodic acid Schi! (PAS) staining method. Although some pinocytosis of polymers may take place, the cytotoxicity is not increased. The morphological changes were relatively small compared to those observed in in vitro cytotoxicity tests of other biomaterials like crosslinked dermal sheep collagen [29,30] and hardly increased after 24 h. In the following two days, con#uent cell layers were formed, which showed that there were no additional growth inhibitory e!ects after 24 h. The observed cell growth inhibition in the presence of dextran or dex-MA was not caused by an osmotic e!ect of these relatively concentrated polymer solutions, since the osmolality of the medium (about 370 mOsm/kg) was only increased with 10}20 mOsm/kg by addition of these polymers. The possible contribution of methacrylic acid (MAA), formed by the hydrolysis of dex-MA and dex-HEMA, to the cytotoxic e!ect was investigated. To study the

possible catalysis of medium components on hydrolysis of the methacrylate ester, dex-MA was incubated with bu!ered medium. After three days, the released amount of hydrolyzed MAA measured corresponded well with the concentration calculated using the reaction rate constant k of the hydrolysis of dex-MA at 373C at pH 7.4,  i.e. 80 and 440 g/ml MAA for dex-MA DS 5 and DS 30, respectively [24]. This demonstrates that the medium components do not catalyze the degradation. The cytotoxicity of MAA was tested in two relevant concentrations, i.e. 105 and 470 g/ml, which is slightly higher than the estimated amount of MAA formed in 72 h at pH 7.4 by dex-MA polymers with a DS of 5 and 30, respectively. The low concentration of MAA (105 g/ml) did hardly exert any cytotoxicity (Fig. 2). At 470 g/ml MAA the CPII was 35$8% (Fig. 2), indicating that MAA has some cytotoxic e!ects at this concentration, although no morphological changes were observed. Comparison of the CPII of MAA at 470 g/ml with that of dex-MA DS 30 (Fig. 2) indicated that the cytotoxicity of this dex-MA polymer solution is lower than expected from additive e!ects of dextran T40 and MAA. This might be due to the gradual release of MAA from dexMA, which is less toxic to cells than a comparable single dose of MAA. 3.3. Hydrogel discs and microspheres Hydrogels, both discs and microspheres, obtained by polymerization of the methacryloyl groups in methacrylated dextrans were extracted extensively to remove possible leachable compounds like residual initiator (KPS) and accelerator (TEMED). Fig. 5 shows that the CPIIs of the hydrogel discs were comparable to the CPIIs of the dex-MA or dex-HEMA polymer solutions depicted in Fig. 2, independent of the DS or the initial water content of the hydrogel discs. After incubation with the hydrogel discs derived from dex-MA DS 5 with an initial water content of 90%, the cells had a normal morphology, apart from some cellular lysis. No intracellular granula or lipid droplets, as observed for the polymer dex-MA, were present. This indicates that these hydrogel discs did not a!ect the "broblasts. However, in cultures incubated with hydrogel discs derived from dex-MA DS 30, some cells next to the gel on one side detached and a small cell-free zone appeared within the "rst 24 h. The detached cells formed aggregates, which adhered to the hydrogel in the next 24 h (data not shown). Apart from these local e!ects, the cell proliferation and morphology were comparable to the control cultures. The adherence of cells to the hydrogel discs with DS 30 might be ascribed to the presence of a high number of hydrophobic domains on the gel, due to the methacrylate esters [6]. Since the conversion rate after radical polymerization is usually above 95% [21,31], the cytotoxicity of the

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Fig. 5. The cell proliferation inhibition index (mean%$SD) after 72 h for di!erent dextran hydrogel discs and microspheres. The initial water content (%) and the DS of the methacrylate-derivatized dextran is depicted. Data are from a representative experiment, performed in triplicate.

small amount of MAA, released from the hydrogels by hydrolysis of residual non-crosslinked MA-groups, will be negligible. In contrast to the hydrogel discs, the hydrogel microspheres gave no or only a slight inhibition of cell proliferation (0}20$2%; the highest value for dexlactate-HEMA DS 4 microspheres; Fig. 5). The morphology of the cells could not be monitored during the 3 days' incubation period due to the non-transparent layer of microspheres on top of the cells. At the end of the culturing period, no morphological deviancies compared to control cultures were observed. The di!erence in cytotoxicity of the hydrogel discs, especially of those with high DS, with that of the microsphere preparations may be explained by a combination of mechanical stress, higher hydrophobicity of the discs with DS 30, and possibly by low concentrations of leachables still present in the discs, because of their poorer extractability compared to that of the small microspheres. Furthermore, di!erences in preparation methods of microspheres and discs may also be responsible for the observed cytotoxicity, although being still acceptable, of the rigid hydrogel discs. In summary, based on the relatively small cytotoxic e!ects of dextran-based hydrogels determined with this sensitive in vitro test, together with the good biocompatibility of hydrogel discs observed in vivo [19], we conclude that these methacrylate-derivatized dextran hydrogels are biocompatible and promising biomaterials for drug delivery purposes.

investigation was supported by the Dutch Technology Foundation (STW), research grant UFA 55.3931.

References
[1] Talmadge JE. The pharmaceutics and delivery of therapeutic polypeptides and proteins. Adv Drug Del Rev 1993;10:247}99. [2] Lee VHL, Yamamoto A. Penetration and enzymatic barriers to peptide and protein absorption. Adv Drug Del Rev 1990; 4:171}207. [3] Park H, Park K. Biocompatibility issues of implantable drug delivery systems. Pharm Res 1996;13:1770}6. [4] Coleman DL, Gregonis DE, Andrade JD. Blood}materials interactions: the minimum interfacial free energy and the optimum polar/apolar ratio hypotheses. J Biomed Mater Res 1982; 16:381}98. [5] Ratner BD. In: Williams DF, editor. Biocompatibility of clinical implant materials, vol. II. Boca Raton, FL: CRC Press, 1981 [chapter 7]. [6] Smetana Jr K. Cell biology of hydrogels. Biomaterials 1993; 14:1046}50. [7] Van Dijk-Wolthuis WNE, Hoogeboom JAM, Van Steenbergen MJ, Tsang SKY, Hennink WE. Degradation and release behavior of dextran-based hydrogels. Macromolecules 1997;30:4639}45. [8] Hennink WE, Franssen O, Van Dijk-Wolthuis WNE, Talsma H. Dextran hydrogels for the controlled release of proteins. J Control Rel 1997;48:107}14. [9] Franssen O, Vandervennet L, Roders P, Hennink WE. Degradable dextran hydrogels: controlled release of a model protein from cylinders and microspheres. J Control Rel 1999;60:211}21. [10] Reynolds JEF, editor. In: Martindale, The extra pharmacopoeia, 31st ed. London: The Pharmaceutical Press, 1996. p. 760}2. [11] Larsen C. Dextran prodrugs * structure and stability in relation to therapeutic activity. Adv Drug Del Rev 1989;3:103}54. [12] Edman P, Ekman B, Sjo K holm I. Immobilization of proteins in microspheres of biodegradable polyacryldextran. J Pharm Sci 1980;69:838}42. [13] Br+ndstedt H, Hovgaard L, Simonsen L. Dextran hydrogels for colon-speci"c drug delivery. II. Synthesis and characterization. Eur J Pharm Biopharm 1995;41:341}5. [14] Shukla AJ. In: Wade A, Weller PJ, editors. Handbook of pharmaceutical excipients, 2nd ed. London: The Pharmaceutical London, 1994. p. 362}6.

Acknowledgements We would like to thank Dr. P.B. Van Wachem for stimulating discussions, E.H. Blaauw and M.J. Van Steenbergen for expert technical assistance, and R.J.H. Stenekes for providing the dex-HEMA polymers. This

C.J. De Groot et al. / Biomaterials 22 (2001) 1197 } 1203 [15] Chirila TV, Thompson DE, Constable IJ. In vitro cytotoxicity of melanized poly(2-hydroxyethyl methacrylate) hydrogels, a novel class of ocular biomaterials. J Biomater Sci Polym Edn 1992;3:481}98. [16] CmH fkova H I, Brynda E, Mandys V, Stol M. Irritation e!ects of residual products derived from p(HEMA) gels. II. Compounds extracted from hydrogels. Biomaterials 1988;9:372}5. [17] Chirila TV, Walker LN, Constable IJ, Thompson DE, Barrett GD. Cytotoxic e!ects of residual chemicals from polymeric biomaterials for arti"cial soft intraocular lenses. J Cataract Refract Surg 1991;17:154}62. [18] Quinn CP, Pathak CP, Heller A, Hubbell JA. Photo-crosslinked copolymers of 2-hydroxyethyl methacrylate, poly(ethylene glycol) tetra-acrylate and ethylene dimethacrylate for improving biocompatibility of biosensors. Biomaterials 1995;16:389}96. [19] Cade H e JA, Van Luyn MJA, Van Wachem PB, Brouwer LA, De Groot CJ, Den Otter W, Hennink WE. In vivo biocompatibility of dextran-based hydrogels. J Biomed Mater Res 2000;50: 397}404. [20] Biological evaluation of medical devices-part 5: tests for cytotoxicity: in vitro methods, ISO/DIS 10993-5(EN 30993-5). International Organisation of Standardisation, Geneva, 1992. [21] Van Dijk-Wolthuis WNE, Franssen O, Talsma H, Van Steenbergen MJ, Kettenes-Van den Bosch JJ, Hennink WE. Synthesis, characterization and polymerization of glycidyl methacrylate derivatized dextran. Macromolecules 1995;28:6317}22. [22] Van Dijk-Wolthuis WNE, Tsang SKY, Kettenes-Van den Bosch JJ, Hennink WE. A new class of polymerizable dextrans with hydrolyzable groups: hydroxyethyl methacrylated dextran with and without oligolactate spacer. Polymer 1997;38:6235}42.

1203

[23] Van Dijk-Wolthuis WNE, Kettenes-Van den Bosch JJ, Van der Kerk-Van Hoof A, Hennink WE. Reaction of dextran with glycidyl methacrylate: an unexpected transesteri"cation. Macromolecules 1997;30:3411}3. [24] Van Dijk-Wolthuis WNE, Van Steenbergen MJ, Underberg WJM, Hennink WE. Degradation kinetics of methacrylated dextrans in aqueous solution. J Pharm Sci 1997;86:413}7. [25] Stenekes RJH, Franssen O, Van Bommel EMG, Crommelin DJA, Hennink WE. The preparation of dextran microspheres in an all-aqueous system: e!ect of the formulation parameters on particle characteristics. Pharm Res 1998;15:557}61. [26] Franssen O, Hennink WE. A novel preparation method for polymeric microparticles without the use of organic solvents. Int J Pharm 1998;168:1}7. [27] Edmundson IC. Particle size analysis, vol. 2. In: Advances in Pharmaceutical Sciences, London: Academic Press, 1967. p. 95}179. [28] Choksakulnimitr S, Masuda S, Tokuda H, Takakura Y, Hashida M. In vitro cytotoxicity of macromolecules in di!erent cell culture systems. J Control Rel 1995;34:233}41. [29] Van Luyn MJA, Van Wachem PB, Olde Damink LHH, Dijkstra PJ, Feijen J, Nieuwenhuis P. Relations between in vitro cytotoxicity and crosslinked dermal sheep collagen. J Biomed Mater Res 1992;26:1091}110. [30] Van Luyn MJA, Van Wachem PB, Nieuwenhuis P, Jonkman MF. Cytotoxicity testing of wound dressings using methylcellulose cell culture. Biomaterials 1992;13:267}75. [31] Stenekes RJH, Hennink WE. Polymerization kinetics of dextranbound methacrylate in an aqueous two phase system. Polymer 2000;41:5563}9.