Abstract
BACKGROUND:Polylactic acid (PLA) surface is hydrophobic and there are no natural recognition sites, so its application is limited. Many different strategies have been studied such as composition and chemical grafting, to produce hydrophilic groups. However, these traditional methods are always involved in complex process and many organic reagents, resulting in decrease of biocompatibility.
OBJECTIVE: To conduct the surface modification of PLA and improve its hydrophilicity with low temperature plasma treatment.
DESIGN: Controlled observation.
SETTING: Department of Materials Science and Engineering in Jinan University.
MATERIALS: The experiment was processed from October 2004 to October 2005 at Guangzhou Research Center of Artificial Organs and Materials Engineering (Ministry of Education). The mainly used materials were: Poly-D, L-lactic acid (Mr 2.9×104, Shandong Medical Instrument Institute), N-vinyl-pyrrolidone (NVP, Acros Company, purified before use).
METHODS: The materials were treated in the plasma reactor under different conditions (power=150 W, t =3, 2, 1 minutes; power=30 W, t = 10, 8, 5, 3, 1 minutes) to choose the optimal condition, and then PLA were grafted with NVP by gas phase polymerization and liquid polymerization, respectively.
MAIN OUTCOME MEASURES: The surface morphology of the films was observed with scanning electron microscopy and atomic force microscopy. The hydrophilicity of native and treated material surfaces was measured using a water contact angle meter. Surface composition was detected with Fourier transform infrared spectrometer. Mass changes measurement was applied to characterize the grafting efficiency.
RESULTS: Scanning electron microscopy and atomic force microscopy showed that the plasma modified materials possessed coarse surface with pores and notches. Water contact angles decreased obviously from 78° to 50° after grafting. Fourier transform infrared spectrometer showed a new absorption peak at 1 637.19 cm-1, which corresponded to the carbonyl stretch vibration absorption of amide group in poly-N-vinyl-pyrrolidone. After the plasma treatment at low temperature, the mass change ratio of PLA film material was -0.6.
CONCLUSION: The plasma treatment and polymerization can improve the hydrophilicity and cause coarse surface of PLA material, which will be helpful for the cell attachment.



INTRODUCTION

Polymers are frequently used as biomaterials in areas of tissue engineering such as tissue replacement, tissue reinforcement and organ transplantation[1-5]. Polylactic acid (PLA) is one of the few synthetic biodegradable polymers, and is widely applied as sutures and orthopedic devices, for which high mechanical strength and toughness are required. However, it is hydrophobic and there are no natural recognition sites on the surface of PLA[6]. The surface hydrophobicity and low surface energy will affect cell adhesion on the PLA, which is critical because cell adhesion occurs before other events like cell spreading, cell migration and differentiation[7-8]. It has been reported that the cell adhesion to a substrate involves the following steps: adsorption of serum proteins on the substrate, contact of round cells to the substrate, attachment of the cells to the substrate and the spreading of the cells on the substrate[9]. Therefore, the surface properties of the materials are very important for the adhesion of proteins, and it is commonly accepted that the adhesion of cells to solid substrate is influenced by several substrate surface properties, such as hydrophilicity, surface charge, roughness and topography.
Plasma treatment is an effective and widely used method for modifying the surface of materials. It has been reported that the surface of cell culture devices, such as petri dishes, micro-carriers and membranes, can be modified by plasma treatment to improve cell adhesion[10-13]. An important feature of plasma treatment is that it affects only the surface of a material subjected to treatment and a very thin near-surface layer whose thickness varies from 100 ? to several micrometers. The bulk of the polymer remains intact under these conditions, retaining the mechanical, physicochemical, and electro-physical properties of the original material surfaces. As a rule, the improvement of adhesive properties by plasma treatment is caused by not only surface cleaning of contaminants but also the formation of hydrophilic groups differing in their chemical nature, which provide high adhesive properties of the modified surfaces[14].
N-vinyl-pyrrolidone (NVP) possesses active double bond, which can be initiated easily to produce its polymer poly-N-vinyl-pyrrolidone (PVP). PVP as a kind of water soluble polymer, has been applied widely in fields of medicine, cosmetics, vintage and drink, etc[15-16]. In this study, we tried to functionalize the surface of poly-D, L-lactic acid (PDLLA) film with plasma treatment and graft polymerization to improve its hydrophilicity.

EXPERIMENTS AND CHARACTERIZATION

Plasma treatment and graft polymerization on PDLLA film surface
A RF plasma graft polymerizer at low temperature

(VTC-FSN-200, Japan, 13.56 MHz) was used for the surface modification. PDLLA films were placed in the glass reactor with a diameter of 5 cm and a length of 20 cm, and then a vacuum pump was connected. When the pressure got to 3 Pa, argon gas was input to increase the pressure to 20 Pa. Afterwards the 13.56 MHz RF source supplied the input power to the plasma reactor. The materials were treated with following parameters respectively: Wf=150 W, t =3, 2, 1 minutes; Wf=30 W, t =10, 8, 5, 3, 1 minutes.
Surface grafting polymerization was then carried out in two methods.①Gas phase polymerization: NVP was input to the reactor under vacuum after plasma treatment. The gas state NVP reacted with films in reactor for certain minutes (1, 2 and 3 minutes);②Liquid polymerization: PDLLA films were taken out from the reactor after plasma treatment and then immersed into NVP water solution (at 0.1, 0.2, 0.3 and 0.4 mass fractions) under N2 atmosphere with shaking for 12 hours.
The products were washed with distilled water to remove NVP monomer and homopolymer PVP and then dried at 60 ℃.

Characterization and analysis of the modified PDLLA films
The surface morphology of the films was observed with scanning electron microscopy (SEM, JSM-T300, Japan) and atomic force microscopy (AFM, CP-Research, USA). The water contact angle of native and treated material surfaces was measured using a Contact Angle Meter (CAM-PLUS, USA) at room temperature. Surface composition was measured with Fourier transform infrared spectrometer (FTIR, Bruke EQUINOX 55, Germany). Mass changes measurement was applied to characterize the grafting efficiency.

RESULTS

Surface morphology observation
Figure 1 shows the surface morphology of PDLLA films observed by SEM, which revealed that plasma had erosion effects on films.

Figure 1a is the surface of plasma treated PDLLA film with low power (30 W, 3 minutes), and Figure 1b shows the surface morphology of the films which were grafted with PVP. The obvious pores and notches on both surfaces were visible. The toughness of the surface will be helpful for the cell attachment[17].
Figure 2a is the AFM images of micro area on the surface. Figure 2b is produced by computer. Many micro pores, juts and notching with different size (up to 10 μm) were seen, which were formed during the film preparation and plasma treatment.

Surface water contact angle measurement
PDLLA films were hydrophobic with water contact angle (θ) of 78°. After plasma treatment, the value decreased to 10° (Table 1). Due to the plasma treatment, plenty of free radical such as -OH, -COOH and -C=O were produced on the PDLLA film surface, which improved the polarity of the films, therefore improved the hydrophilicity and decreased the water contact angle.

With high power 150 W, time affected the contact angle slightly, which ranged from 20° to 45°. θvalue changed greatly for each sample because of the coarse surface of the materials. To avoid the damage of high power to materials, low power 30 W was studied as well. It was very interesting that contact angle decreased much more. For some samples, the contact angle decreased even to 0°. Similarly, time affected the results not so much.
According to the contact angle, 30 W was preferred to 150 W for reaction. With higher power, more corrosion was caused, and on the other hand, many polar groups were removed as well, and then caused the decrease of the polarity and increase of the water contact angle.
The water contact angel of the treated films increased with time and recovered to about 70° two weeks later. Therefore plasma polymerization was applied, which were expected to improve the hydrophilicity.
Plasma polymerization will make up for these defects and modify the surface of materials eternally. According to above results, the PDLLA films were first treated with plasma (Wf=30 W, t =3 minutes), and then with the newly produced active free radials on the surface to initiate polymerization of NVP. The polymerization was carried out in gas phase method and liquid method respectively.
The effects of grafting polymerization by gas method on surface contact angle were studied first. The results showed that θ value decreased from 78° to about 50° after polymerization. But time affected not so much on the surface hydrophilicity, and after polymerization of 1, 2 and 3 minutes, the contact angles were 50-55, 48-52 and 47-53, respectively.
Comparing to gas method, liquid method had less effects on surface water contact angle of PDLLA film (Table 2). Modified with 0.2 mass fraction of NVP solution, contact angle decreased to 53°-55°, which was close to the value of gas method. Either lower or higher concentration of NVP solution could not decrease the value more. However, liquid method is easier to be operated. Therefore, polymerization with 0.2 mass fraction of NVP would be the best method for plasma surface modification of PDLLA films.

FTIR spectroscopy
FTIR spectra of the plasma treated PDLLA films and PVP grafted PDLLA films are shown in Figure 3. The absorption of -C-H of PLA was at 2 992.73 cm-1. 1 451.85 cm-1 and 1 367.32 cm-1 were the absorption of -CH3 bending vibrations. Strong peak of 1 751.25 cm-1 was from -C=O of ester. Comparing with both spectra (Figure 3a) and (Figure 3b), there was a new weak peak at 1 637.19 cm-1 on NVP grafted PDLLA films, which was from -C=O of amide group. The result revealed that PVP was grafted on the surface of PDLLA film.

Mass change of PDLLA films
The mass change of the PDLLA was calculated with the following equation: M%= (M-M0)/M0×100%
M0 is the original mass of films, and M is the mass of films after grafting polymerization.

M% was used to characterize indirectly the grafting efficiency of the polymerization. In this study, only liquid polymerization was studied because of its more merits than gas method. Figure 4 demonstrates that after plasma treatment, the mass of PDLLA films decreased with the M% of -0.6, because plasma damaged the materials surface (Figure 1), even though there were newly produced groups. The mass increased obviously after the films were reacted with NVP solution. NVP solution at 0.2 mass fraction had highest M%, which was in accordance with the results of Table 2. The higher concentration (0.3 and 0.4 mass fraction) of NVP solution dissolved the materials in some degrees, and then caused the mass decrease of the films.

DISCUSSION

There are plenty of electrons, ions and neutral ions in plasma, which will transfer the energy to the atoms or molecules of the materials surface when they contact with the materials and then generate different physical and chemical changes. Some particles can also be implanted to the material surface to cause collision, scattering, re-arrangement or crystallize, etc. and change the original surface properties. Therefore, after plasma treatment, PLA surface presented many micro pores and juts. In addition, due to the plasma treatment, plenty of free radicals such as -OH, -COOH and -C=O were produced on the PDLLA film surface, which improved the polarity of the films, therefore improved the hydrophilicity and decreased the water contact angle. According to the contact angle, 30 W was preferred to 150 W for reaction. With higher power, more corrosion was caused, and on the other hand, many polar groups were removed as well, and then caused the decrease of the polarity and increase of the water contact angle.
The water contact angel of the treated films increased with time and recovered to about 70° two weeks later. There are many reasons for such phenomena, i.e. polar groups reacted with each other, or active groups in surrounding etc. Yasuda thought that the main reason is that the new produced polar groups turned to below the surface of the materials[18]. Plasma polymerization will make up for these defects and modify the surface of materials eternally[19-20].
According to above results, the PDLLA films were first treated with plasma (Wf=30 W, t =3 minutes), and then with the newly produced active free radial on the surface to initiate polymerization of NVP. The polymerization was carried out in gas method and liquid method, respectively.
NVP was able to dissolve PLA to some degrees, which removed the newly produced active polar groups. Meantime polymerization and interaction between the active groups will get to a balance during certain period and terminate the reactions. Although gas grafting method could improve the hydrophilicity obviously, it has many disadvantages. It wastes much solvent during the process, and it is not quantitative when the reactors are concerned. Similarly, as liquid method, high concentration of NVP could dissolve more PLA surface. NVP polymerization at 0.2 mass fraction would be the best method for plasma surface modification of PDLLA films.

CONCLUSION

Plasma modification of PDLLA films was studied in this experiment. Water contact angle measurement showed that the hydrophilicity of the films was improved much after surface plasma treatment even at lower power (30 W) and in less time (3 minutes), but it recovered to original level two weeks later. Plasma graft polymerization of NVP could modify the material eternally, which was carried out in gas method and liquid method. Although both methods decrease the contact angle greatly, liquid method with NVP solution at 0.2 mass fraction is preferable considering the operation and accuracy of reaction. Furthermore, plasma treatment and polymerization may cause coarse surface, which will be helpful for the cell attach. Its more physiochemical properties and biocompatibility will be studied in the future.

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聚乳酸膜等离子接枝聚合聚乙烯基
吡咯烷酮及表面性能研究**☆

李立华，丁 珊， 田 冶，田金环，周长忍
暨南大学科学与工程系，广东省广州市 510630
李立华☆，女，1977年生，山东省德州市人，汉族，暨南大学在读博士，讲师，主要从事组织工程及相关生物材料的研究。
通讯作者：周长忍，教授，博士，暨南大学材料科学与工程系，广东省广州市 510630
国家自然科学基金资助（30170272）*；广东省重大专项基金资助（A302020104）*
摘要
背景：由于聚乳酸表面亲水性差、缺乏天然分子识别位点等缺点而限制了其应用。通过复合、化学接枝等方法试图引进亲水性基团，但过程比较复杂，并应用大量有机试剂，影响材料的生物相容性。
目的：利用低温等离子技术对聚乳酸进行表面改性，改善其亲水性。
设计：对比观察。
单位：暨南大学科学与工程系。
材料：实验于2004-10/2005-10在广州人工器官和材料工程研究中心实验室完成。实验用主要材料：聚乳酸（Mr 29 000，山东医疗器械研究所），乙烯基吡咯烷酮(美国Acros公司，使用前经重蒸纯化)。
方法：通过改变不同的工作条件(功率=150 W，时间=3，2，l min；功率=30 W，t=10，8，5，3，l min) 选择优化条件，然后通过气相法和常压液相法进行PLA-乙烯基吡咯烷酮接枝反应。
主要观察指标：通过扫描电镜和原子力显微镜观察材料的表面形态；水接触角测量仪测定材料等离子处理和等离子接枝前后亲水性的变化；傅里叶变换红外光谱仪测定材料的结构变化；通过材料改性前后质量变化分析材料的接枝情况。
结果：①扫描电镜显示等离子处理和接枝后的材料表面呈现大小不一的气孔和沟痕。②接枝后的水接触角由原来的78°下降到50°。③傅里叶变换红外光谱图表征1 750 cm-1吸收峰的低波数侧1 637.19 cm-1出现新的吸收峰，该峰对应于聚乙烯基吡咯烷酮链段上酰胺基团中的羰基伸缩振动吸收。④聚乳酸膜材料经低温等离子处理后，材料的质量变化百分数为-0.6。
结论：等离子处理和接枝后材料的亲水性有明显变化，材料表面呈现大小不一的气孔和沟痕，此表面有利于细胞的黏附。
关键词：聚乳酸；乙烯基吡咯烷酮；等离子；接枝聚合
中图分类号: R318.08 文献标识码: A 文章编号: 1673-8225(2008)14-02757-04
李立华，周长忍.聚乳酸膜等离子接枝聚合聚乙烯基吡咯烷酮及表面性能研究[J].中国组织工程研究与临床康复，2008，12(14):2757-2760
[www.zglckf.com/zglckf/ejournal/upfiles/12-14/14k-2757(ps).pdf]
(Edited by: Huang YC/Yang Y/Wang L)