Subscriber access provided by ZONGULDAK KARAELMAS UNIV Biomacromolecules is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Article Synthesis and Characterization of Polymeric Soybean Oil-g-Methyl Methacrylate (and n-Butyl Methacrylate) Graft Copolymers: Biocompatibility and Bacterial Adhesion Birten akmakl, Baki Hazer, shak zel Tekin, and Fsun Beendik Cmert Biomacromolecules, 2005, 6 (3), 1750-1758• DOI: 10.1021/bm050063f • Publication Date (Web): 25 March 2005 Downloaded from http://pubs.acs.org on April 25, 2009 More About This Article Additional resources and features associated with this article are available within the HTML version: • Supporting Information • Links to the 3 articles that cite this article, as of the time of this article download • Access to high resolution figures • Links to articles and content related to this article • Copyright permission to reproduce figures and/or text from this article
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Subscriber access provided by ZONGULDAK KARAELMAS UNIV
Biomacromolecules is published by the American Chemical Society. 1155 SixteenthStreet N.W., Washington, DC 20036
Article
Synthesis and Characterization of Polymeric SoybeanOil-g-Methyl Methacrylate (and n-Butyl Methacrylate) Graft
Copolymers: Biocompatibility and Bacterial AdhesionBirten akmakl, Baki Hazer, shak zel Tekin, and Fsun Beendik Cmert
Biomacromolecules, 2005, 6 (3), 1750-1758• DOI: 10.1021/bm050063f • Publication Date (Web): 25 March 2005
Downloaded from http://pubs.acs.org on April 25, 2009
More About This Article
Additional resources and features associated with this article are available within the HTML version:
• Supporting Information• Links to the 3 articles that cite this article, as of the time of this article download• Access to high resolution figures• Links to articles and content related to this article• Copyright permission to reproduce figures and/or text from this article
Department of Chemistry, Faculty of Arts and Sciences, and Departments of Immunology and MedicalMicrobiology, Faculty of Medicine, Zonguldak Karaelmas University, 67100 Zonguldak, Turkey
Received January 27, 2005
Peroxidation, epoxidation, and/or perepoxidation reactions of soybean oil under air at room temperatureresulted in cross-linked polymeric soybean oil peroxides on the surface along with the waxy soluble part,sPSB, with a molecular weight of 4690, containing up to 2.3 wt % peroxide. This soluble polymeric oilperoxide, sPSB, initiated the free radical polymerization of either methyl methacrylate (MMA) orn-butylmethacrylate (nBMA) to give PSB-g-PMMA and PSB-g-PnBMA graft copolymers. The polymers obtainedwere characterized by1H NMR, thermogravimetric analysis, differential scanning calorimetry, and gelpermeation chromatography techniques. Polymeric oil as a plasticizer lowered the glass transition of thePSB-g-PMMA graft copolymers. PSB-g-PMMA and PSB-g-PnBMA graft copolymer film samples werealso used in cell culture studies.Fibroblastandmacrophage cellswere strongly adhered and spread on thecopolymer film surfaces, which is important in tissue engineering. Bacterial adhesion on PSB-g-PMMAgraft copolymer was also studied. BothStaphylococcus epidermidisand Escherichia coliadhered on thegraft copolymer better than on homo-PMMA. Furthermore, the latter adhered much better than the former.
Introduction
Much attention has been paid to studying and developingenvironmentally biodegradable plastics to retard or eradicateplastic pollution.1,2 Current interest in cheap biodegradablepolymeric materials has encouraged the development of suchmaterials from readily available, renewable, inexpensivenatural sources such as starch, polysaccharides, and edibleoils.3 Today natural oils and fats are considered to be themost important class of renewable resources for the produc-tion of biodegradable polymers in two ways. The first is theproduction of poly(3-hydroxyalkanoate)s (PHAs) as anenergy reserve material by some microorganisms by usingplant oils and fish oils.4 The second is direct polymerizationof the oils, for example, a copolymerization with divinyl-benzene and styrene, leading to thermoset copolymers,5 orpolymerization of vinyl,6 maleic anhydride,7 glycidyl ether,8
and norbornyl9 derivatives of the oil. Our efforts haverecently focused on grafting reactions of monomers onnaturally occurring peroxidized polymeric drying oils suchas linseed oil.10 Oxidation of linseed oil in the air involveshydrogen abstraction from a methylene group between twodouble bonds in a polyunsaturated fatty acid chain.11-13 Thisleads to peroxidation, perepoxidation, hydroperoxidation,epoxidation, and then cross-linking via radical recombination.
Soybean oil is a triglyceride with two dominant fatty acidresidues, linoleic acid and oleic acid, and an average numberof double bonds per molecule of 4.6. The average MW ofsoybean oil is around 874, and the oil contains linoleic acid(51%), oleic acid (25%), palmitic acid (11%), linolenic acid(9%), and stearic acid (4%) residues.14,15Scheme 1 indicatesthe structure of soybean oil.
Poly(methyl methacrylate) (PMMA) is used widely inmedical practices, especially in intraocular lenses and bonecement. Biodegradable plastics are also of interest for medicalapplications because of their biocompatibility. The celladhesion and spreading on a surface are the most indicativeprocesses to assess the biocompatibility of a syntheticpolymer.16 Because of their strong ability to adhere ondifferent polymeric surfaces, L-929 fibroblast cells are themain cell type widely used in biocompatibility studies.Macrophages are important components of the mammalianimmune system as professional antigen-presenting cells andnonspecific killers of a wide variety of pathogens. In vivouse of synthetic and biologically derived polymers inbiomedical applications such as tissue engineering and drugdelivery introduces an interaction with the host immunesystem that can determine the efficacy of the particularapplication.17
In many biomedical applications the adhesion of bacteriato biomaterials causes undesirable inflammation or infection.Bacterial adherence to polymer surfaces varied significantlydepending on the polymer type as well as the strain of thebacteria. In recent years various groups have thereforefocused on the development of bioinert, biocompatiblecoatings which can be used to minimize protein adsorption
* To whom correspondence should be addressed. Phone: 011 90 372322 17 03. Fax: 011 90 372 323 86 93. E-mail: [email protected],[email protected].
† Department of Chemistry, Faculty of Arts and Sciences.‡ Department of Immunology, Faculty of Medicine.§ Department of Medical Microbiology, Faculty of Medicine.
and bacterial adhesion while maintaining the mechanical andphysical properties of the underlying substrate.18
In this work, as a cheap and abundant renewable source,soybean oil was converted to polymeric peroxide underatmospheric conditions at room temperature. Then it wasused to initiate the graft copolymerization of MMA orn-butylmethacrylate (nBMA) to obtain a new biodegradable mate-rial. These newly synthesized graft copolymers are testedfor their biocompatibilities and effects on bacterial adhe-sion.19
Experimental Section
Materials. Soybean oil was locally supplied and used asreceived. MMA and nBMA were supplied from Aldrich and
freed from inhibitor by vacuum distillation over CaH2. Allother chemicals were reagent grade and used as received.
Formation of Polymeric Soybean Oil under LaboratoryConditions. For the formation of polymeric soybean oil, 5.0g of soybean oil spread out in a Petri dish (L ) 16 cm) wasexposed to sunlight in the air at room temperature. After agiven time, a gel polymer film associated with a waxy andviscous liquid was formed. Chloroform extraction of thecrude polymeric oil for 24 h at room temperature allowedseparation of the soluble part of the polymeric soybean oil(sPSB) from the gel (gPSB). The results and conditions ofpolymer formation from soybean oil are listed in Table 1.Peroxygen contents of the sPSB samples varied from 1.3 to
Scheme 1. Chemical Structure of Soybean Oil
Table 1. Results and Conditions of the Polymerization and the Peroxidization of Soybean Oil
Synthesis of PSB-g-PMMA and PSB-g-PnBMA Copolymers Biomacromolecules, Vol. 6, No. 3, 2005 1751
2.3 wt %. TheMw of sPSB sample 60-4 was 4690 withMWD ) 1.52.
Peroxygen Analysis.Peroxide analysis of soluble PSBfractions was carried out by refluxing a mixture of 2-propanol(50 mL)/acetic acid (10 mL)/saturated aqueous solution ofKI (1 mL) and 0.1 g of the polymeric sample for 10 minand titrating the released iodine against thiosulfate solutionaccording to the literature.20 Peroxide contents of the solublepolymeric soybean oil fractions are listed in Table 1.
Graft Copolymerization. For graft copolymerization ofPSB peroxides with a vinyl monomer, a given amount ofPSB and methyl methacrylate orn-butyl methacrylate werecharged separately into a Pyrex tube. Argon was introducedthrough a needle into the tube for about 3 min to expel theair. The tightly capped tube was then put into a water bathat 80 °C. After the required time, the contents of the tubewere coagulated in methanol. The graft copolymer sampleswere dried overnight under vacuum at 30°C. The charac-teristic data for MMA and nBMA copolymerization initiatedby the PSB peroxides are listed in Table 2.
Fractional Precipitation of the Graft Copolymers. In atypical fractional precipitation procedure,21a-c 0.5 g of
polymer sample was dissolved in 10 mL of CHCl3. Methanolwas used as a nonsolvent and kept in a 50 mL buret.Afterward methanol was added to the polymer solution withcontinuous stirring, until the polymer began to precipitate.At this point, theγ value was calculated by taking the volumeratio of the nonsolvent (methanol) consumed to the solvent(chloroform, 10 mL).
Polymer Characterization. 1H NMR spectra were re-corded in CDCl3 at 17°C with a tetramethylsilane internalstandard using a 400 MHz NMR AC 400 L.
The molecular weight of the polymeric samples wasdetermined by gel permeation chromatography (GPC) witha Waters model 6000A solvent delivery system with a model401 refractive index detector and a mode 730 data moduleand with two Ultrastyragel linear columns in series. Chlo-roform was used in the elution at a flow rate of 1.0 mL min-1.A calibration curve was generated with polystyrene standards.
Differential scanning calorimetry (DSC) thermogramswere obtained on a Netzsch DSC 204 with a CC 200 liquidnitrogen cooling system to determine the glass transitiontemperatures (Tg), and thermogravimetric analysis (TGA) of
Table 2. Results and Conditions for the Polymerization of MMA and nBMA Initiated by PSB (Sample 60-4 in Table 1) at 80 °C
a Calculated from their 1H NMR spectra (by comparison with peaks at 3.7 ppm (PMMA) and 4.2 ppm (PSB)).
Figure 1. 1H NMR spectrum of the PSB sample (no. 60-4 in Table1). (The characteristic peaks of the precurser soybean oil have beenmarked on the spectrum.)
Figure 2. 1H NMR spectrum of the PSB-g-PMMA block copolymersample (56-6).
1752 Biomacromolecules, Vol. 6, No. 3, 2005 Cakmaklı et al.
the polymers obtained was performed on a PL TGA 1500instrument to determine thermal degradation. For DSCanalysis, samples were heated from 20 to 200°C at a rateof 10 °C/min (first heating) and held at the final temperaturefor 1 min to eliminate the thermal history applied to thesamples. After being cooled to-100 °C, they were thenreheated to 200°C at a rate of 10°C/min (second heating).
Cell Culture and Cell Adhesion Studies.The murinefibroblast cell line (L-929) and macrophage cell line (RAW264.7) were purchased from the American Type CultureCollection (ATCC; Rockville, MD). The cell culture stocksolution (RPMI-1640), which contains 10% (v/v) heat-inactivated fetal bovine serum with 100 units/mL penicillinand 100µg/mL streptomycin, was supplied from Gibco.
In a typical cell culture study, a Petri dish (L ) 60 mm)was coated with the graft copolymer film (thickness∼1 mm)by means of chloroform solution casting. The polymer-film-coated Petri dish was sterilized by using ethylene oxide. A6 mL sample of cell culture containing 0.8× 105/mL L-929(or 0.5× 105/mL RAW 264.7) was poured into the polymer-coated Petri dish and incubated at 37°C in humidified aircontaining 5% (v/v) CO2. For a given time, the cells on thepolymer surface were observed with an inverted microscope(Nikon Eclipse TE 300, Tokyo, Japan) and photographedwith a Minolta Dimage 7i camera (magnification 200×). Thecell photographs taken with this camera associated with theabove microscope are given in Figures 7 and 8.
Bacterial Adherence. One Staphylococcus epidermidisstrain and oneEscherichia colistrain obtained from twodifferent patients who had infections related to intravascularcatheters were used for the adherence tests. The bacteria werekept at -80 °C in skim milk. A 10 µL sample of thebacterium culture was inoculated onto a blood agar plate(Oxoid, U.K.; tryptone (14.0 g/L), peptone (4.5 g/L), yeastextract (4.5 g/L), sodium chloride (5.0 g/L), agar (12.5 g/L),and sheep blood (7 wt %)) and kept overnight at 37°C.Bacterial suspensions of 108 colony-forming units (CFUs)/mL were prepared for each bacterium for the adherence testsaccording to the method cited in ref 22.
Method: A polymer disk (thickness∼1 mm,L ) 6 mm)was placed under sterile conditions in 1 mL of bacterialsuspension and incubated at 37°C for 30 min. The polymerdisk was removed and rinsed with 2 mL of sterile phosphate-buffered solution (PBS) three times for 60 s to eliminatenonadhering bacteria. The polymer disk was transferred into1 mL of PBS in a glass tube and agitated for 3 min via vortexat 2400 rpm/min. A 10µL sample of PBS containingdislodged bacteria was seeded onto blood agar plates and
Figure 3. 1H NMR spectrum of the PSB-g-PnBMA block copolymersample (62-3).
Table 3. Thermal Analysis Results of the Graft Copolymers andthe Related Homopolymers
Figure 4. GPC chromatograms of the fractionally precipitated PSB-g-PMMA and PSB-g-PBMA graft copolymers.
Synthesis of PSB-g-PMMA and PSB-g-PnBMA Copolymers Biomacromolecules, Vol. 6, No. 3, 2005 1753
spread to facilitate subsequent colony counting. Ten-folddilutions were made to calculate an accurate count of bacteriaadhered to the polymer disk surfaces. Ten-fold-dilutedcolonies were counted by the naked eye after 24 h ofincubation at 37°C. The bacterial density per polymer type((CFUs/mL)/mm2) was calculated by dividing the colonynumber mean by the total surface area (mm2) of the polymerdisk.
Results and Discussion
Polymeric Soybean Oil Containing Peroxide Groups.Polymeric soybean oil containing peroxide/hydroperoxidegroups was obtained in the air at room temperature for eightweeks. PSB samples contained cross-linked films associatedwith the waxy soluble part (sPSB), which was isolated withthe chloroform extraction of the cross-linked film. Theviscous soluble part formed under the cross-linked film onthe surface can also be separated easily. As expected, thecross-linked soybean oil amount increases to 38 wt % at 150days of polymerization time. The results and conditions ofthe peroxidized polymeric soybean oil are listed in Table 1.The molecular weight of sample 60-4 was 4690 with MWD) 1.52, and peroxygen contents were found to be between1.3 and 2.3 wt %. Figure 1 shows the1H NMR spectrum ofthe PSB sample (no. 64 in Table 1). The characteristic peaksof the precurser soybean oil have been marked on thespectrum which confirms the PSB segments in the copolymerstructure.
Graft Copolymerization. Because of their peroxidegroups, sPSB samples initiated the copolymerization ofMMA or nBMA at 80 °C to obtain PMMA-g-PSB andPnBMA-g-PSB in high yield. Copolymerization conditionsand copolymer analysis results are listed in Table 2. Thehigher concentration of PSB in monomer solution yields ahigher amount of cross-linked graft copolymer except forruns 56-6 and 62-4. Interestingly, the highest concentrationof PSB feeding (ca. 3.00 g of PSB) yields a lower amountof cross-linked copolymer. Cross-linked and soluble graftcopolymer fractions were isolated by means of chloroformextraction.
Soluble fractions of the graft copolymers were fractionallyprecipitated to determine theγ values of the graft copolymersand their related homopolymers. Homo-PMMA and homo-PnBMA were precipitated in theγ ranges 3.0-3.8 and 2.8-4.1, respectively, while PSB-g-PMMA and PSB-g-PnBMAcopolymer fractions were precipitated in theγ ranges 2.5-3.8 and 2.5-3.9, respectively. Becauseγ values of the graftcopolymers and related homopolymers were almost super-imposed, fractional precipitation was useful only to determinethe γ values of oil-grafted copolymers instead of to isolatepure graft copolymers from the related homopolymers. Aswe discuss below, unimodal GPC curves can be attributedto the pure graft copolymers freed from the related ho-mopolymers. Homo-sPSB, a pale yellow viscous liquid, wasalready eliminated by staying in the solution during theprecipitation procedure.
1H NMR spectra of the soluble copolymer samples of PSB-g-PMMA (run 56-6,γ ) 2.0-4.0) contained characteristic
peaks as indicated in Figure 2 (δ, ppm): -COOCH3 ofMMA at 3.7 and-CH2- of SB at 2.8, 2.4, 1.9, 1.4, and0.9; the peaks at 4.1-4.4 ppm originate from the protons inthe methylene groups of the triglyceride. The vinylic protonsare detected at 5.3 ppm. For this sample, PSB inclusion wasfound to be 12 mol % by taking the ratio of the signals at3.7 and 4.2 ppm. PSB inclusion of the graft copolymers isproportionally increased with the PSB macroinitiator in thefeed (compare runs 56-2, -5, and -6 in Table 2).
1H NMR spectra of the soluble copolymer samples of PSB-g-PnBMA (run 62-3,γ ) 2.0-4.0) contained characteristicpeaks as indicated in Figure 3 (δ, ppm): -COOCH3 ofnBMA at 4.0 (shifted to higher field than that of PMMA)and-CH2- of SB at 2.8, 2.4, 1.9, 1.4, and 0.9; the peaks at
Figure 5. DSC traces of PSB-g-PMMA (runs 56-2, 56-6, and 56-5)and PSB-g-PnBMA (runs 62-2 and 62-3).
Figure 6. Thermogravimetric traces of PSB, PnBMA, and PSB-g-PMMA (run 56-2) and PSB-g-PnBMA (run 62-3) graft copolymers.
1754 Biomacromolecules, Vol. 6, No. 3, 2005 Cakmaklı et al.
4.1-4.4 ppm originate from the protons in the methylenegroups of the triglyceride (which partially overlapped withthe peak of PnBMA at 4.0 ppm). The vinylic protons aredetected at 5.3 ppm.
GPC was used to determine the molecular weights andpolydispersity of the copolymers. Fractionated samples ofPSB-g-PMMA and PSB-g-PnBMA gave unimodal traceswhich can be attributed to the graft copolymer structurewithout homopolymer impurities (see Figure 4).
Thermal analysis of graft copolymers was performed byDSC and TGA. Table 3 lists the glass transition (Tg), meltingtransition (Tm), and decomposition (Td) temperatures. Figure5 indicates the thermogravimetric traces of sPSB and solublePMMA-g-PSB graft copolymers.
A considerable plastization effect of polymeric oil in thePMMA graft copolymers has been observed by lowering theglass transition to 83°C compared with 110°C for homo-PMMA. There is no big difference in the case of PnBMA
graft copolymers.Tg values of the graft copolymers werearound of that of homo-PnBMA. Figure 5 also indicates DSCtraces of the polymers.Tg values of PSB were shifted tohigher values in PMMA graft copolymers, while they werenot observed in PnBMA copolymers because the aliphaticside chain of PnBMA may help the compatibilization ofpolymeric oil. Figure 6 indicates the thermogravimetric tracesof sPSB, PnBMA, and soluble PnBMA-g-PSB graft copoly-mers. The first temperature region at around 130-280 °C(stage I) is related to evaporation and decomposition of theunreacted free oil in the bulk polymer. It is interesting thatthe TGA traces of the graft copolymers were in the middleof the related homopolymers. When we compare theTd
values of homo-PMMA and homo-PnBMA with theTd
values of the graft copolymer of soybean oil, the latter areshifted to higher values (see Table 3).
Cell Culture and Adhesion.We have chosen L-929 cellsas the fibroblast and RAW 264.7 cells as the macrophage
Figure 7. L-929 cell growth and adhesion test on the polymer samples: (a) 1 h, (b) 16 h.
Synthesis of PSB-g-PMMA and PSB-g-PnBMA Copolymers Biomacromolecules, Vol. 6, No. 3, 2005 1755
cell line. Figures 7 and 8 show L-929 fibroblast and RAW264.7 macrophage cell adhesion and proliferation on homo-copolymers and graft copolymers, respectively. PSB-g-
PMMA and PSB-g-PnBMA film samples, PMMA- andPnBMA-coated Petri dishes, and a standard PS Petri dishset for the fibroblast cell culture and 62-2- and 62-3-coded
Figure 8. RAW 264.7 growth and adhesion test on the polymer samples: (a) 1 h, (b) 16 h.
1756 Biomacromolecules, Vol. 6, No. 3, 2005 Cakmaklı et al.
copolymers, PMMA- and PnBMA-coated Petri dishes, anda standard PS Petri dish set for the macrophage cell culturewere used. There were abundant spherical cells at thebeginning in all dishes (Figures 7a and 8a). In the first hour,L-929 cells in the copolymer 56-5 dish were adheredsignificantly better than the other cells, and they turned intotheir authentic shapes, except RAW cells on PnBMA. Thecell adhesion ability of RAW macrophages in PnBMA graftcopolymer (62-3) was found to be greater than that of theothers (Figure 8). These results were similar to those of thestandard PS dish.
After 16 h of incubation, the amounts of adhered L-929(Figure 7b) and RAW (Figure 8b) cells were significantlyincreased for all polymeric surfaces. At this time, the amountsof adhered L-929 cells in 56-5, 62-2, and 62-3 were foundto be greater than those of the others. The RAW cells didnot have their own authentic shape on the PnBMA surfacefor 16 h. It is important that PnBMA-g-PSB is suitable formacrophage growing and adhering while homo-PnBMA isnot suitable. However, the macrophage cell adhesion abilityin 62-3 was found to be greater than that of the others. Itwas also observed that the macrophage cell adhesion abilityin 62-2 was found to be higher than that of PnBMA. Thesefindings were similar to those of standard PS Petri dish usageand supported the biocompatibility of the graft copolymersobtained.
Bacterial Adhesion.Bacterial adhesion on any surface isnecessary for development of infection. Despite significantadvances, bacterial adhesion to polymer surfaces is still asignificant problem. There are many studies involved inbacterial adhesion on different polymer surfaces.23-25
In this study, the adherence of bacteria to copolymerPMMAs was compared with that of PMMA. While theadherences ofS. epidermidisand E. coli to PMMA diskswere similar, a significant decrease in the adherence ofE.coli to the PSB-PMMA graft was observed. The greatestdecrease (1/324) in the adherence ofE. coli was observedwith 56-5 (Table 4). The adherence was 34 and 48 timeslower in 56-2 and 56-6, respectively. Although the decreasein the adherence ofS. epidermidiswas far less than that ofE. coli, there was a 1.1-, 1.6-, and 1.4-fold reduction inadherence to PSB-g-PMMA graft copolymers 56-2, 56-5,and 56-6, respectively. More significant reduction of adhe-sion was observed withE. coli, especially to polymer 56-5.Similary, Tunney et al. reported adhesion of the hydrophilicE. coli isolates to the copolymers increased with decreasingcopolymer hydrophobicity. A relationship was not apparentbetween copolymer hydrophobicity and adherence of the
hydrophobicEnterococcus faecalisisolate in their study.26
The development of surfaces that reduce adherence ofbacteria may have several applications, for instance onmedical devices used in the urogenital tract, where catheter-associated infections are rampant andE. coli constitutes themost important causative organism for infection. Accordingto our results PSB-g-PMMA is a promising novel copolymerof PMMA for use in medical devices, with the advantage ofdecreasing bacterial adherence.
Conclusion
Naturally peroxidized polymeric soybean oil as a macro-initiator initiates the free radical polymerization of MMAand nBMA, without any additional catalyst, which may beimportant for medical applications of the graft copolymers.Soybean oil inclusion acts as a plasticizer and can makePMMA and PnBMA partially biodegradable and biocom-patible. Fibroblast and macrophage cells strongly adheredon the graft copolymers, especially PSB-g-PnBMA. Bacterialadherence to PSB-g-PMMA was found to be lower than thatto PMMA.
Acknowledgment. This work was financially supportedby the Zonguldak Karaelmas University Research Fund.
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Table 4. Adherence of Bacteria to PMMA and PSB-g-PMMADetermined by Direct Counting of Viable Adherent BacteriaReleased by Vortex Agitation ((CFUs/mL)/mm2)a
polymer S. epidermidis E. coli
PMMA 46428 42857PSB-g-PMMA
56-2 39285 125056-5 28214 13256-6 31321 892
a The bacterial density ((CFUs/mL)/mm2) (CFUs ) colony-forming units)was calculated by dividing the colony number mean by the total surfacearea of the polymer disk.
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