Biocompatibility of Phema and P(Hema-Co-Sma) Hydrogels

Transcription

1 Key Engineering Materials Online: ISSN: , Vols , pp doi: / Journal Citation (to be inserted by the publisher) Copyright 2005 Trans by Trans Tech Publications, Tech Publications Switzerland Biocompatibility of Phema and P(Hema-Co-Sma) Hydrogels Bo Hyung Park 1, Young A Han 2, Jin Hyun Choi 3 and Jeong Ok Lim 2* 1 Department of Medical and Biological Engineering, Kyungpook National University, Daegu, , Korea 2 Medical Research Institute, Kyungpook National University, Daegu, , Korea 3 Department of Natural Fiber Science, Kyungpook National University, Daegu, , Korea * Corresponding author : jolim@knu.ac.kr Keywords: PHEMA, P(HEMA-co-SMA), mechanically strong hydrogel, cytotoxicity, cell adhesion, implantation, in vivo tissue reaction, biomaterials, biocompatibility. Abstract. To replace a poly(2-hydroxyethyl methacrylate) (PHEMA) sponge, which has limited applications as an implant material, PHEMA and poly(2-hydroxyethyl methacrylate-co-sodium methacrylate) (P(HEMA-co-SMA)) hydrogels with enhanced biocompatibility were prepared based on the copolymerization of 2-hydroxyethyl methacrylate (HEMA) and sodium methacrylate (SMA) at a high monomer. When the cytotoxicity, cell adhesion, and in vivo tissue reaction of the resulting hydrogels were investigated, the results suggest that hydrogels prepared by the copolymerization of HEMA and SMA at a high monomer have great potential as implant materials with an excellent biocompatibility. Introduction Poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogels have already been utilized as biocompatible synthetic materials for some time. Depending on the of HEMA in the water, PHEMA hydrogels with different properties can be synthesized. For example, a sponge-like hydrogel can be prepared with a below 55-60% [1]. However, a soft PHEMA sponge is not so strong mechanically and only has limited applications, such as reconstructive breast surgery and nasal cartilage replacement [2-3]. Moreover, after the implantation of a PHEMA sponge, macrophage formation has been reported [4]. Conversely, a mechanically strong hydrogel can be obtained with a higher monomer, yet it has a low water content. Therefore, to solve these problems, the current study prepared PHEMA and copolymer hydrogels with an increased mechanical strength and water content based on using a high monomer and a hydrophilic comonomer, sodium methacrylate (SMA). The cytotoxicity, cell adhesion, and in vivo tissue reaction of the synthesized PHEMA and P(HEMA-co-SMA) hydrogels were then investigated to determine the influence of SMA on the biological properties of the hydrogels. Materials and Methods Synthesis of PHEMA and P(HEMA-co-SMA) hydrogels. The PHEMA hydrogels were synthesized by the free radical polymerization of HEMA and ethylene glycol dimethacrylate (EGDMA) in water. The desired molar ratio of HEMA ( mol/l water ) and EGDMA ( mol/mol EGDMA ) was mixed with ammonium persulfate ( mol/mol HEMA ) in distilled water, then tetramethylethylenediamine ( mol/mol HEMA ) was added to the mixture. The polymerization was carried out at room temperature for 24 hrs. The P(HEMA-co-SMA) hydrogels were also synthesized using the same method as described above. The total monomer of HEMA and SMA was mol/l water and the relative molar ratios of SMA to HEMA were 0.01 and 0.05 [Table 1]. After polymerization, the hydrogels were immersed in an ethanol/deionized water mixture for 24 hrs and washed repeatedly with deionized water. All chemicals mentioned above were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). All rights reserved. No part of contents of this paper may be reproduced or transmitted in any form or by any means without the written permission of Trans Tech Publications, (# , Pennsylvania State University, University Park, USA-18/09/16,05:44:12)

2 52 On the Convergence of Bio-, Information-, Enrivonmental-, Energy-, Spaceand (to Nano-Technolgies be inserted by the Title of Publication publisher) Cytotoxicity assay (MTT assay). The cytotoxic effects were evaluated indirectly by quantifying the mitochondrial dehydrogenase activity via the enzymatic conversion of MTT tetrazolium to a colored formazan product. Since the reduction of MTT only occurs in metabolically active cells, the level of activity is a measure of cell viability. As such, an MTT colorimetric assay is a routine cytotoxicity test that is useful for the preliminary screening of biomaterial toxicity [5-6]. The control group of ROS17/2.8 cells and experimental group of ROS17/2.8 cells including the hydrogels were incubated for 24 h at 37. MTT (250 μg / ml ) was then added to the cultures and the cells incubated for another 4 h at 37. After aspiration of the medium, dimethyl sulfoxide (DMSO) was used (150 μl /well, 24-well plate) to solubilize the dark purple formazan compound formed during the incubation. The samples were read at 570 nm. All chemicals mentioned above were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Cell adhesion. ROS17/2.8 osteoblasts and C2C12 myoblasts were used to assess the cell adhesion to the hydrogels. The cells were seeded on the hydrogels at a density of cells/well (24-well plate) and left at 37 for 72 hrs. After washing with phosphate buffered saline (PBS), the samples were fixed with 2 % formaldehyde and observed under an optical microscope ( 100). In vivo tissue reaction. To estimate the in vivo biocompatibility of the hydrogels, male Sprague-Dawley rats (body weight, g) were used. The PHEMA hydrogel was implanted subcutaneously (s.c.) into the lower back, while the P(HEMA-co-SMA) (SMA feed ratio: 0.01, 0.05) hydrogels were implanted (s.c.) below the left and right side of the neck, respectively (Fig. 1). After 1 or 8 weeks, the tissue surrounding the hydrogels was stained with hematoxylin and eosin (H & E). Statistical analysis. The results of the MTT assay were expressed as the mean ±S.D. values, and the statistical significance of the results was determined by an unpaired t-test (*p<0.05, **p<0.01). Results and Discussion The growth and metabolism of cells with biomaterials depend on the hydrophilic/hydrophobic character of the comonomers and chemical structure of the hydrogels. Cell viability. An MTT assay was used to evaluate the effects of PHEMA and P(HEMA-co-SMA) hydrogels on cell viability, the results of which are shown in Figure 2. The mean absorbance of the control group was (94±7) 10-2, while the mean absorbances of the P(HEMA-co-SMA) hydrogels with SMA feed ratios of 0, 0.01, and 0.05 were (43±18) 10-2, (64±15) 10-2, and (89±7) 10-2, respectively. As such, it was found that the cell viability increased relative to the SMA feed mole ratio (0.05>0.01>0). The cell viability with the PHEMA hydrogel was significantly low than that of the normal cell culture plate. However, when incorporating the SMA moiety, the cell viability was enhanced remarkably. Hence, the cytotoxicity of the PHEMA hydrogel was seemingly minimized by copolymerization with SMA. One possible explanation is that methacrylates or acrylates with hydroxy groups, like HEMA, have a relatively high cytotoxicity [7]. Cell adhesion. Cell adhesion depends on the hydrophilicity and electrostatic properties of the hydrogel. As such, the adhesion and spreading of the osteoblast and myoblast cells on the surface of the P(HEMA-co-SMA hydrogel prepared with an SMA feed mole ratio of 0.01 were the best, as shown in Figure 3. While, the P(HEMA-co-SMA) hydrogel prepared with an SMA feed ratio of 0.05 exhibited a lower cell adhesion than the PHEMA hydrogel, implying that cell adhesion was inhibited when the negative charge of the SMA moiety surpassed the stimulation by hydrophilicity [8-14]. In vivo biocompatibility. One and 8 weeks after implantation, the histological findings for the tissue surrounding the PHEMA hydrogel revealed the formation of multinucleated giant cells and fibrosis, while no evidence of significant foreign body reactions was found in the tissue around the P(HEMA-co-SMA) hydrogels [Fig. 4].

3 Key Engineering Materials Vols Title of Publication (to be inserted by the publisher) Therefore, the current results indicate that the P(HEMA-co-SMA) hydrogels with an appropriate hydrophilic SMA molar ratio provided increased cell viability, good cell adhesive characteristics, and good biocompatibility. Table 1. Preparation conditions for PHEMA and P(HEMA-co-SMA) hydrogels Monomer feed ratio (mol SMA / mol HEMA ) Crosslinking agent APS TMEDA c a b Fig. 1. Implantation of PHEMA (a) P(HEMA-co-SMA) (SMA feed mole ratio: 0.01) (b) P(HEMA-co-SMA) (SMA feed mole ratio: 0.05) (c) hydrogels 1. 2 Absorbance (570 nm) * * * * c o ntro l Monomer feed ratio (mol SMA /m ol HEMA ) Fig. 2. Results of MTT cell viability assay of hydrogels. Each column and vertical bar shows mean± S.D. (control; cell culture plate, 0; PHEMA hydrogel, 0.01; P(HEMA-co -SMA) hydrogel (SMA feed mole ratio: 0.01), 0.05; P(HEMA-co-SMA) hydrogel (SMA feed mole ratio: 0.05))

4 54 On the Convergence of Bio-, Information-, Enrivonmental-, Energy-, Spaceand Title of Publication (tonano-technolgies be inserted by the publisher) R O S 17/2.8 C2C 12 (A) (B ) (C ) Fig. 3. Cell adhesion to PHEMA (A), P(HEMA-co-SMA) (SMA feed mole ratio: 0.01) (B), P(HEMA-co-SMA) (SMA feed mole ratio: 0.05) (C) hydrogels. Observation under optical microscope, magnification w eek k 8 w eeks (A) (B) (C). Fig. 4. Histology of rat hypodermic tissue at PHEMA hydrogel-implanted site (A) P(HEMA-co-SMA) (SMA feed mole ratio: 0.01) hydrogel-implanted site (B) and P(HEMA-co-SMA) (SMA feed mole ratio: 0.05) hydrogel-implanted site (C). Observation under optical microscope, magnification 100

5 Key Engineering Materials Vols Title of Publication (to be inserted by the publisher) Summary Poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogels have already been utilized as biocompatible synthetic materials. However, PHEMA is not mechanically strong and has a low water content. Accordingly, to solve these problems, the current study prepared PHEMA and P(HEMA-co-SMA) hydrogels with an increased mechanical strength and water content using a high monomer and hydrophilic comonomer, such as sodium methacrylate (SMA). When the cytotoxicity, cell adhesion, and tissue compatibility of the resulting hydrogels were investigated, it was concluded that the hydrogels prepared by the copolymerization of HEMA and SMA at a high monomer provided increased cell viability, good cell adhesive characteristics, and good biocompatibility. References [1] T. C. Warren and W. Prins: Macromolecules Vol. 5 (1972), p [2] J. S. Calnan, J. J. Pflug, A. S. Chhabra and N. Raghupati: Br. J. Plast. Surg. Vol. 24 (1971), p. 113 [3] Z. Voldrich, Z. Tomanek, J. Vacik and J. Kopecek: J. Biomed. Mat. Res. Vol. 9 (1975), p [4] S. Vijayasekaran, J. H. Fitton, C. R. Hicks, T.V. Chirila, G. H. Crawford and I. J. Constable: Biomaterials Vol. 19 (1998), p [5] F. Rosso, A. Barbarisi, M. Barbarisi, O. Petillo, S. Margarucci, A. Calarco and G. Peluso: Materials Science and Engineering C Vol. 23 (2003), p [6] S. Alavez, D. Pedroza, J. Moran: Neurochemistry International Vol. 43 (2003), p [7] E. Yoshii: J. Biomed. Mat. Res. Vol. 37 (1997), p [8] J. H. Lee, J. W. Lee, G. Khang and H. B. Lee: Biomaterials Vol. 18 (1997), p [9] L. Werner, S. K. Pandey, M. Escobar-Gomez: Ophthalmology Vol. 107 (2000), p [10] L. Werner, S. K. Pandey, D. J. Apple: Ophthalmology Vol. 108 (2001), p [11] M. R. Bet, G. Goissis, S. Vargas and H. S. Selistre-de-Araujo: Biomaterials Vol. 24 (2003), p [12] P. B. van Wachem, A. H. Hogt, T. Beugeling, J. Feijen, A. Bantjes, J. P. Detmers and W. G. van Aken: Biomaterials Vol. 8 (1987), p [13] Q. Qiu, M. Sayer, M. Kawaja, X. Shen and J.E. Davies: J. Biomed. Mat. Res. Vol. 42 (1998), p [14] T. A. Horbett, J. J. Waldburger, B. D. Ratner and A. S. Hoffman: J. Biomed. Mat. Res. Vol. 22 (1988), p. 383.