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Tsinghua Science and Technology, Vol. 6, No. 3, August 2001 pp. 216-221 Effect of Surface Modification on Microbiol Polyhydroxyalkanoate Films on Biocompatibility YANG Xianshuang Department of Biological Sciences and Biotechnology, Tsinghua University,
Beijing 100084, China; * Supported by the Tsinghua University “985” Project Fund Received: 2000-08-31 Code Number: ts01069 Abstract: The purpose of this study was to investigate in vitro biocompatibility of a new type of polymer, polyhydroxybutyrate-co-hexanoate (PHBHH x). The hydrop hilicity and biocompatibility were studied with two kinds of enzymes, amylase BA N480L and lipase Novozym388. The degree of hydrophilicity was observed using con tact angle measurements. in vitro biocompatibility evaluations were carried out by direct incubation of mouse fibroblast cell line L929 on the polyhydroxyalkano ate (PHA) films. The sa mples treated with BAN480L showed that the PHA biocompatibility increased while the hydrophilicity decreased. Relative to untreated samples, the number of cells on the Novozym388 modified PHBHHx significant decrease as the hydrophilicity al so decreased. The results indicated that other surface characteristics besides h ydrophilicity influence the biocompatibility of PHBHHx films. Key words: polyhydroxybutyrate-co-hexanoate (PHBHHx); hydrophilicity; biocomp atibility Introduction Polyhydroxyalkanoates, abbreviated as PHA, is a family of biopolyesters synthe si zed by many organisms[1, 2] , with over 100 different types based on the length and composition of the carbon chain. PHA behavior is similar to that of conventi onal plastics such as polyethylene and polypropylene except that they are biodeg radable; therefore, there have been many research projects to develop PHA into a biodegradable plastic[3, 4]. However, the high production cost of PHA h as proh ibited its wide application as a “plastic”. In addition to their biodegradabi li ty, PHA can also be used as biomaterials for medical purposes because of their wide range of mechanical properties, since cost is not a major concern with biom aterials[5]. One biomaterial application for PHA is in the area of tissue engineering. In thi s case, biocompatibility is the most important property that biomaterials must m eet, since cell proliferation and tissue formation are dependent on the interact ion between the cells and the matrix[6]. If PHA is to be employed as a m atrix for g rowing real tissues, its biocompatibility should be carefully evaluated through a series of biocompability tests. In vitro, biocompatibility is mainly evaluated by measuring the cell adhesion and cell proliferation on the biomaterial. Biocompatibility of implants is determined by the degree of fibrotic and inflammatory reaction surrounding the samples[7]. With in vitro testing, the bio material surface characteristics may have an important influence on its biocompatibility. The material surface characteristics may have to be modified to suit the intende d application, often without altering the other properties of the matrix, such a s its mechanical strength or thermal properties. Potential surface modifications include changes in chemical group functionality, surface charge, hydrophilicity , hydrophobicity, and wettability. It has been reported that pH treatment can liberate acid groups or charged species, which promote cellular attachment and cell proliferation[8,9]. A polymer surface can be modified by various chemical or physical processes including plasma-ion beam treatment, electric discharge surface grafting, chemical reactions, vapor deposition of metals and flame treatment [8]. In this paper, the effect of enzymes on the hydrophilicity of one kind of PHA, polyhydroxybutyrate-co-hexanoate (PHBHHx), was determined by measuring the contact angles[10]. The biocompatibility of differently treated PHA poly mers was evalu ated by observing their effects on the proliferation of the mouse fibroblast cell li ne L929 measured by MTT [3-(4,5-dimethylthiazol-2-yl)-diphenyl-tetraz olium bromide] [11-13]. 1 Materials and Methods 1.1 PHA materials and its purification PHBHHx were provided by Guangdong Jiangmen Center for Biotech Development and th e Procter & Gamble Co., Cincinnati, U.S.A. The PHBHHx was dissolved in acetone and refluxed at 60 °C for one hour. The PHBHH x acetone solution was then centrifuged to remove non-soluble particles (5000 g, 20 min). Methanol was added to the supernatant to obtain high purity PHBHHx pre cipitates. The PHB was already 99.99% pure and was not subjected to further purification. 1.2 PHA film casting The purified PHA was dissolved in chloroform, then poured into petri dishes. The dishes were kept at room temperature to allow complete evaporation of the chloroform. The evaporation of the solvent resulted in the formation of PHA films on the petri dishes. Vacuum drying was then used to completely remove any possible solvent remaining in the films as the solvent is toxic to the cell lines and may influence the results. Before cell line inocul ation, the films were sterilized under ultraviolet radiation overnight. 1.3 PHA film surface modification using lipases BAN480L and Novozym388 were donated by Novo Nordisk China (Beijing, China). The Novozym388 (20 000 LU) was diluted to generate 2000, 4000, 6000, 8000, an d 10 000 LU solutions at pH 7.5. The BAN480L was diluted to 1, 2, 3, 4, and 5 fold a t pH 7.5. The PHA films were immersed in each of the lipase solutions and incuba ted at 35 °C and pH 7.5 for one hour, with occasional gentle shaking. The hydrophilicity of the PHA films was measured in terms of the conta ct angle using a contact angle measurement facility (Beijing Analytical Instrume nt Co.[5], Ltd.) BT(2+1*3 1.4 Effect of PHBHHx polymer on the prolifer ation of L929 HTH 1.4.1 Cells and cell cultureSTWTHT The mouse fibroblast cell line L929 was purchased from the Institute of Virology Academia Sinica. The cell line was cultured in 50 mL polystyrene flasks incubat ed in a CO2 incubator supplied with 5% CO2 at 37 °C . The culture medium was Dulb ecco's modified eagle medium (DMEM) supplemented with 10% fetal calf serum and 1 ㏑ penicillin-streptocin solution. After the cell line grew to completely cover the 50 mL cell culture flask, 5 mL of DMEM containing 0.01% trypsinase were added to the culture flask to digest the interconnected cells. 1.4.2 Growth of the cell line on PHBHHx films The mouse fibroflast cells were grown to confluence followed by detachment of t he cells using 1% sterile trypsin in DMEM without serum. The cell concentration was determined with a hemocytometer. A cell suspension containing 5x104 cells per millilitre was added to the PHA film in t he petri dishes with the same concentration of cells cultured on untreated PHBHH x films as a control. The petri dishes were then incubated for 72 h. Cell morpho l ogical examinations in every dish were performed daily using an inverted phase c ontract microscope to observe the cell growth including spreading, attachment an d death. 1.4.3 In vitro biocompatibility evaluation After incubation for 72 h, the culture media were discarded and the petri dis h es were rinsed with phosphate buffered saline (PBS, pH7.4 ) to remove the ser um i n the media. Five millilitres of serum-free medium and 1 mL of MTT solution wer e added to eac h sample. The staining reagent was prepared using dimethylthiazol-2-yl-diphen yl-tetrazolium bromide (MTT) (Sigma, USA) dissolved at a concentration of 5 mg /mL in sterile phosphate-buffered saline (PBS, pH 7.4) added with 0.05% glucose. The s olution was passed through a 0.22 mm filter to remove any formazan crystals and t he filtrate was stored at -20 °C in the dark[14,15]. The dish es were all incubated for another 3 hour at 37 °C . Then, the medium and MTT were replaced b y 5 mL of is opropanol solution containing 10% formic acid[16] , which was incubated for ano ther 20 minutes at room temperature. During this period, the yellow MTT solution was converted to insoluble blue formazan crystals by the mitochondrial dehydrog enase in the living cells. The crystals were then dissolved in 10 mL isoprop anol with the optical density of this solution then immediately measured using a spe ctrophotometer at 550 nm[17]. Theoretically, the intensity of the observ ed blue color is proportional to the cell number/metabolic activity[ㄠㄧ] . Three paral lel replicates were measured for each sample with the average taken as the final result. An MTT assay was performed to count the L929 cells; with the absorbance plotted against the counted number of cells. Then, the concentration of viable cells on the dish bottom was determined by the solution absorbance. 1.5 Scanning electron microscopy (SEM) The PHB and PHBHHx films, including the untreated and lipase modified samples , were observed using a scanning electron microscopy. 1.6 Statistical analysis The data was expressed as the average of three points and analyzed using Micr osoft Excel. The figures were drawn with Microsoft Word and Microsoft Excel. 2 Results 2.1 Correlation between MTT assay and direct cell counting The MTT was performed on directly counted mouse fibroblast L929 cells, with cell densities of 10x105, 5x105, 2.5x105, 1.25x105, 6.25x104, 3.12x1 04, and 1.56x104 (mL-1 ). Linear regression analysis of the results sh owed a linear correlation b etween the number of directly counted cells and the MTT absorbency at 550 nm, y= 0.040 +2.517x10-6 x, r2=0.987 (Fig.1). The number of cell s on the PHA films in the following experiments was determined using the calibration curve. 2.2 Effects of enzymes on the hydrophilicity of the PHBHHx films The hydrophilicity of PHBHHx surfaces treated with BAN480L and Novozym 388 was studied by measuring the contact angles of the treated films. As shown in Fig.2, the contact angle of the PHBHHx film decreased from 8 9.4° to 66.7° as the BAN480L concentration increased, with the lowest contact angle at an enzyme concentration of 20%. Lipase Novozym388 had a more significant effect on the PHBHHx film with the contact angle again decreasing as the Novozym388 con centration increased. After treated with the enzyme at 50% of the original enzym e concentration, the contact angle decreased from 79.5° to 31.9° (Fig.3). Thes e results indicated that both enzymes significantly affected the PHBHHx film surfa ce, so that when used at the proper conditions, they could greatly improve the h ydrophilicity of the treated surface. The enzymes probably transformed the ester bonds or glycosidic bands on the poly mers into hydroxyl groups. The hydroxylation introduced polar groups into the PH BHHx surface and simultaneously roughened the surface with the corruption of the covalent bonds. Both Novozym388 and BAN480L had a dose-dependent effect on the treated surface, although the effect of Novozym388 was much more remarkable than that of BAN480L. With BAN480L, the maximum hydrophilicity occurred at 20% conce ntration rather than 30%, suggesting that the polymer surface structures had bee n broken so that the hydrophilicity decreased when treated with 30% BAN480L. 2.3 Effect of PHBHHx surface modification on the proliferation of cell line L929 The influence of PHBHHx surface modification on cell proliferation was studied b y growing cell line L929 on the lipase treated polymer surfaces. The cell prolif eration was detected by MTT assay. The MTT assay showed that treating with BAN480L could greatly improve L929 cell growth and proliferation on the PHBHHx surface (Fig.4). On PHBHHX treated with 20% BAN480L, the L929 cells achieved the highest density, increasing from 4.8x1 04 to 1.3x105 cells/mL. This result was consistent with the hydrophilicity evalua tion result (Fig.2). Novozym388 treatment also affected the biocompatibility of the PHBHHx surface, but the effect was negative. The cell numbers in Fig.5 did not change much with Novozym388 concentration but were much lower on the modifie d PHBHHx surface than on the untreated sample. 2.4 Effect of lipase on the blended PHA poly mer biocompatibility 2.4.1 Cell viability The cell proliferation results on the blended PHB/PHBHHx polymer films are sh own in Fig.6. The cell densities varied significantly for the different blen ded polymer compositions. The results indicate that the cell densities gradually increased as the PHB decreased and the PHBHHx increased in the blended polymers before treatment by lipase. Cell viability on the modified polymers was better than on the untreated samples. At the same time, the biocompatibility difference s between the various blended polymers decreased with the maximum obtained for c ells grown on the 5:5 PHB/PHBHHx polymer. The results indicate that the lip ase had more influence on the biocompatibility of PHB than PHBHHx. 2.4.2 Scanning electron microscopy (SEM) The surface structures of the PHB films, as seen in Fig.7, show that the PHB film surfaces were unevenly distributed around hollow depressions, the size of which ranged from 1-5 mm, Fig.7(a). These structures were not on the same horizon tal level. After lipase treatment, the hollow depressions disappeared and the PH B surface was transformed into a uniform but coarse surface, Fig.7(b). The surfa ce structures of PHBHHx, Fig.7(c) and lipase modified PHBHHx, Fig.7(d), are different from those for PHB. There was no significant difference between t he two P HBHHx samples, both of which had hollow depressions with sites smaller than 1 m m showing that the lipase had little effect on the change of the PHBHHx surface ch aracteristics. The BAN480L modified PHA films were not observed here due to the limited analysis. 3 Discussion The main aim of this study was to examine the effect of enzymes on the biocompat ibility of PHBHHx, which is a potential biomaterial for tissue engineering. Fact ors such as average relative molecular mass, polydispersity, charge distributi on, resid ual monomers, and the nature of the polymer and its conformation have all been s hown to affect polymer biological properties[18]. The main effects of th e enzym es were to reduce the hydrophilicity and to increase the granulation of the poly mer surface, both of which have an important effect on cell attachment, cell pro liferation, and cell activity. Hydrophilicity is supposed to be one of the most i mportant surface properties for determining biomaterial biocompatibility, as evi denced by cell attachment and proliferation when incubated on the material surface. However, the MTT results showed that the changes with the two enzymes were n ot always consistent. Treatment with increasing concentrations of Novozym388 rem arkably reduced the surface contact angles, which indicated better hydrophilicit y (Fig.4). However, the treated surfaces had much worse biocompatibility than t he control samples (Fig.5). In general, increasing surface hydrophilicity shoul d result in better attachment of the cells to the material surface. Therefore, t hese results suggest that there may be other factors that affect the biocompatib ility of the PHA biomaterial surfaces. The enzymes may change not only the hydro philicity, but also other surface characteristics that are also important for cell proliferation but were not measurable using contact angles. The SEM results (Fig.7) showed that the surface characteristics also influenced the interaction between the cells and the matrix. Lipase treatment of the PHB s ample caused a significant decrease in the porosity and cascade surface structur es, which resulted in much better biocompatibility as measured by cell viability . However, PHBHHx surface characteristics did not change much, which correlates with the small changes in cell viabilities on the PHBHHx samples. The results in dicate that the less porous surface had more useful surface area, which the cell s tended to adhere to. Therefore, other methods that helped flatten the PHA surf ace would improve the polymer's biocompatibility. In summary, PHBHHx is a promising biodegradable polymer for using as a biomateri al in tissue engineering, which has better in vitro biocompatibility than PHB. The contract angle and cell proliferation experiments showed that the hydroxyphilic ity did not correlate positively with the biocompatibility. Lipase and amylase caused some increase of the cell attachment and proliferation on PHA films, but the effect varied with polymer composition. Lipase smoothed the coarse PHB surface which improved cell adhesion. The advantage of a blend of PHA polymers is that the polymer mechanical characteristics vary with composition. The biocompatibili ty of the blend could then be improved by lipase modification. References
Copyright 2001 - Tsinghua Science and Technology The following images related to this document are available:Photo images[ts01069f7.jpg] [ts01069f3.jpg] [ts01069f2.jpg] [ts01069f4.jpg] [ts01069f5.jpg] [ts01069f1.jpg] [ts01069f6.jpg] |
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,
ZHAO Kai
,
XIA Caihong
†,
CHEN Jinchun
,
CHEN Guoqiang 
**
