纳米TiO_2薄膜修饰PMMA IOL的功能探讨
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摘要
目的:对比研究纳米二氧化钛(Titanium Dioxide, TiO_2)光催化剂薄膜修饰聚甲基丙烯酸甲酯(polymethyl methacrylate, PMMA)人工晶状体(intraocular lens, IOL)与未修饰TiO_2薄膜PMMA IOL的表面性能,为今后相关的实验和临床应用提供理论依据。
方法:以主波长为365nm的a波紫外光(ultraviolet a, UVA)作为激发光源(平均光照强度382μW/cm2)。实验分为两个部分,第一部分:①以接触角对样品TiO_2/PMMA IOL进行表征;②细胞黏附实验:将两组晶体UVA预照射不同时间(0、20、40min)后,分别固定于24孔培养板孔内,再接种20μL浓度为2～3×104个/mL的细胞悬液,培养3h后行HE染色观察其形态,并镜下计数IOL表面晶体上皮细胞黏附量;③细胞杀伤实验:细胞同上培养5h后,行TUNEL染色观察形态,并用Image-Pro□Plus图像分析软件评估IOL表面细胞的凋亡率;④计算并分析比较细胞在两组晶体表面的黏附自由能。第二部分:①血小板黏附实验:先采用传统离心法制备富血小板血浆(platelet-rich plasma,PRP),再将两组晶体经UVA预照射不同时间(0、10、30min)后,固定于24孔培养板孔内,每枚接种20μL PRP,培养2h后取出,通过瑞特染色观察其形态,运用软件评估IOL表面血小板的黏附率。②)细菌杀灭实验:两组晶体UVA预照射不同时间(0、10、30min)后,分别置于培养孔后,每孔接种2 mL浓度为1×105cfu/mL的金黄色葡萄球菌和大肠杆菌,培养2h后取出,进行革兰氏染色后普通光学显微镜下观察,用软件评估IOL表面金葡菌的存活率;③分析比较细菌在两组晶体表面的黏附自由能。
结果:第一部分:①随着UVA预照射时间的延长,TiO_2/PMMA IOL表面接触角逐渐减小,亲水性增强,统计学上有显著性差异(P<0.05)。②细胞黏附实验中,UVA预照射不同时间段后,晶体上皮细胞在TiO_2/PMMA IOL表面的黏附量逐渐减少,与PMMA IOL相比差异有统计学意义(P<0.05)。③细胞杀伤实验中,UVA预照20分钟后TiO_2/PMMA IOL表面即可见较多细胞呈凋亡形态,且随预照时间的延长,凋亡率增高,同PMMA IOL相比有显著性差异(P<0.05)。④细胞在TiO_2/PMMA IOL表面黏附自由能△Fadh﹥0,而在PMMA IOL表面的△Fadh<0,从热力学角度可知TiO_2/PMMA IOL表面更不利于细胞黏附。
第二部分: UVA预照射不同时间段后,血小板和细菌在TiO_2/PMMA IOL表面的黏附量均逐渐减少,与PMMA IOL相比差异有统计学意义(P<0.05)。UVA预照射30min后,TiO_2/PMMA IOL表面的血小板黏附率和细菌存活率较低;瑞氏和革兰氏染色后观察到血小板和细菌在TiO_2/PMMA IOL表面都是呈单个散在分布,而在PMMA IOL表面却多数成簇聚集。同样,细菌在TiO_2/PMMA IOL表面黏附自由能△Fadh﹥0,而在PMMA IOL表面的△Fadh<0,从热力学角度可知TiO_2/PMMA IOL表面更不利于细菌黏附。
结论:(1)实验所用TiO_2/PMMA IOL,经过纳米TiO_2薄膜修饰,表面的水接触角变小,亲水性增强;且随着UVA激发时间的延长,TiO_2/PMMA IOL表面水接触角逐渐减小,亲水性逐渐增强。(2)TiO_2/PMMA IOL经UVA激发后,人眼晶体上皮细胞、血小板及金黄色葡萄球菌和大肠杆菌在其表面的黏附与生长受到了明显的抑制。(3)表面黏附自由能计算结果表明,人眼晶体上皮细胞、金黄色葡萄球菌和大肠杆菌在TiO_2/PMMA IOL表面的黏附自由能显著提高,从而不利于黏附。
PURPOSE: To compare the surface property of titanium dioxide (TiO_2) -coated polymethyl methacrylate (PMMA) intraocular lens (IOL) and noncoated PMMA IOLs. This may provide basic information for related study and clinical application in the future.
METHODS: Ultraviolet A (UVA,λ=365nm, 4W, 382μW /cm2) was used as the excitation light source. The study consisted of two parts. Part One: The attribute of TiO_2/PMMA IOL was represented by means of water contact angle; Human lens epithelial cells(HLECs) adhesion test in vitro: after UVA pre-irradiation for different time (0min, 20min, 40min), the IOLs coated or non-coated with Nano-TiO_2 films were cultured with 2～3×104cells/mL of HLECs for 3 hours. At the end of culture, the number of cells adhering to the surfaces of the IOLs was counted under inversion phase microscope; Cell apoptosis test: after UVA illumination for different time(0 min、20 min、40min),cells were cultured for 5 hours, cell morphous was observed by TUNEL staining under ordinary light microscope and the apoptosis rate was evaluated by Image-Pro Plus analysis software. The surface energy components of the various substrates and HLECs were calculated based on Lifshitz-van del Waals/acid-base approach (LW-AB). The surface free energies obtained were used to calculate the interfacial free energies of adhesion (△Fadh ) of cells on various IOLs. Part Two: Platelets adhesion test:after the preparation of platelet rich plasma (PRP) by centrifugation, each IOL with the pre-irradiation for different minutes(0 min、10 min、30min) was cultured with 20μL PRP for 2 hours, then the platelets adhering to the IOL surface were observed with Wright staining and the adhesive rate was assessed by the analysis software; in Sterilization test:after the pre-irradiation of UVA for diffrernt time(0 min、10 min、30min), each IOL was placed under a culture of Staphylococcus aureus (S.A.,1×10~5 colony forming units [CFU]/mL) or Escherichia coli (E.C.BL21, 1×10~5 CFU /mL). After Gram staining, the survival percentage of S.A. was evaluated by software. The interfacial free energies of adhesion (△Fadh ) of two kinds of bacteria on various IOLs was calculated.
RESULTS: The first part of the study showed that as the time of ultraviolet irradiation increased, the contact angle of TiO_2/PMMA IOL grew down apparently(P﹤0.01) comparing with the control group. Cell adhesion test showed that as the pre-irradiation time increased, the number of the adhering cells was significantly lower in the TiO_2-coated IOL group than the noncoated IOL group (P﹤0.01). TUNEL staining showed that most HLECs began apoptosing when exposed to UVA for 20min. The TiO_2/PMMA IOL group had a higher apoptosis rate than PMMA IOL group(P﹤0.01). The second part: As the pre-irradiation time extended, the number of platelets and bacteria sticking on TiO_2/PMMA IOL reduced and the number was notably lesser in the TiO_2-coated IOL group than the noncoated IOL group (P﹤0.01). After UVA pre-irradiation for 30min, platelets and S.A. showed lower adhesive and survival rate on TiO_2/PMMA IOL surface. And Wright and Gram staining showed that both the distribution of platelets and bacteria on TiO_2/PMMA IOL suface were scattered singly, while most were clustered together on the PMMA IOL surface. The facial adhesion free energies of HLECs and bacteria to TiO_2/PMMA IOL were both positive(△Fadh﹥0), while PMMA IOL were negative. So TiO_2/PMMA IOLs would be more unfavourable for cells and bacteria adhesion from the point of view of thermodynamics.
CONCLUSION: (1) The TiO_2/PMMA IOLs coated with nano- TiO_2 thin film were shown to have smaller water contact angle and stronger hydrophilicity. And as the time of UVA irradiation extended, the angle got smaller and the hydrophilicity increased. (2) After UVA irradiation, the adhesion and growth of human lens epithelial cells, platelets, Staphylococcus aureus and Escherichia coli on the surface of TiO_2/PMMA IOL were all suppressed obviously. (3)The computation indicated that the facial adhesion free energy of HLECs, S. aureus and E. coli to TiO_2/PMMA IOLs elevated significantly, resulting an unfavourable intraocular surface for adhesion.
方法:以主波长为365nm的a波紫外光(ultraviolet a, UVA)作为激发光源(平均光照强度382μW/cm2)。实验分为两个部分,第一部分:①以接触角对样品TiO_2/PMMA IOL进行表征;②细胞黏附实验:将两组晶体UVA预照射不同时间(0、20、40min)后,分别固定于24孔培养板孔内,再接种20μL浓度为2～3×104个/mL的细胞悬液,培养3h后行HE染色观察其形态,并镜下计数IOL表面晶体上皮细胞黏附量;③细胞杀伤实验:细胞同上培养5h后,行TUNEL染色观察形态,并用Image-Pro□Plus图像分析软件评估IOL表面细胞的凋亡率;④计算并分析比较细胞在两组晶体表面的黏附自由能。第二部分:①血小板黏附实验:先采用传统离心法制备富血小板血浆(platelet-rich plasma,PRP),再将两组晶体经UVA预照射不同时间(0、10、30min)后,固定于24孔培养板孔内,每枚接种20μL PRP,培养2h后取出,通过瑞特染色观察其形态,运用软件评估IOL表面血小板的黏附率。②)细菌杀灭实验:两组晶体UVA预照射不同时间(0、10、30min)后,分别置于培养孔后,每孔接种2 mL浓度为1×105cfu/mL的金黄色葡萄球菌和大肠杆菌,培养2h后取出,进行革兰氏染色后普通光学显微镜下观察,用软件评估IOL表面金葡菌的存活率;③分析比较细菌在两组晶体表面的黏附自由能。
结果:第一部分:①随着UVA预照射时间的延长,TiO_2/PMMA IOL表面接触角逐渐减小,亲水性增强,统计学上有显著性差异(P<0.05)。②细胞黏附实验中,UVA预照射不同时间段后,晶体上皮细胞在TiO_2/PMMA IOL表面的黏附量逐渐减少,与PMMA IOL相比差异有统计学意义(P<0.05)。③细胞杀伤实验中,UVA预照20分钟后TiO_2/PMMA IOL表面即可见较多细胞呈凋亡形态,且随预照时间的延长,凋亡率增高,同PMMA IOL相比有显著性差异(P<0.05)。④细胞在TiO_2/PMMA IOL表面黏附自由能△Fadh﹥0,而在PMMA IOL表面的△Fadh<0,从热力学角度可知TiO_2/PMMA IOL表面更不利于细胞黏附。
第二部分: UVA预照射不同时间段后,血小板和细菌在TiO_2/PMMA IOL表面的黏附量均逐渐减少,与PMMA IOL相比差异有统计学意义(P<0.05)。UVA预照射30min后,TiO_2/PMMA IOL表面的血小板黏附率和细菌存活率较低;瑞氏和革兰氏染色后观察到血小板和细菌在TiO_2/PMMA IOL表面都是呈单个散在分布,而在PMMA IOL表面却多数成簇聚集。同样,细菌在TiO_2/PMMA IOL表面黏附自由能△Fadh﹥0,而在PMMA IOL表面的△Fadh<0,从热力学角度可知TiO_2/PMMA IOL表面更不利于细菌黏附。
结论:(1)实验所用TiO_2/PMMA IOL,经过纳米TiO_2薄膜修饰,表面的水接触角变小,亲水性增强;且随着UVA激发时间的延长,TiO_2/PMMA IOL表面水接触角逐渐减小,亲水性逐渐增强。(2)TiO_2/PMMA IOL经UVA激发后,人眼晶体上皮细胞、血小板及金黄色葡萄球菌和大肠杆菌在其表面的黏附与生长受到了明显的抑制。(3)表面黏附自由能计算结果表明,人眼晶体上皮细胞、金黄色葡萄球菌和大肠杆菌在TiO_2/PMMA IOL表面的黏附自由能显著提高,从而不利于黏附。
PURPOSE: To compare the surface property of titanium dioxide (TiO_2) -coated polymethyl methacrylate (PMMA) intraocular lens (IOL) and noncoated PMMA IOLs. This may provide basic information for related study and clinical application in the future.
METHODS: Ultraviolet A (UVA,λ=365nm, 4W, 382μW /cm2) was used as the excitation light source. The study consisted of two parts. Part One: The attribute of TiO_2/PMMA IOL was represented by means of water contact angle; Human lens epithelial cells(HLECs) adhesion test in vitro: after UVA pre-irradiation for different time (0min, 20min, 40min), the IOLs coated or non-coated with Nano-TiO_2 films were cultured with 2～3×104cells/mL of HLECs for 3 hours. At the end of culture, the number of cells adhering to the surfaces of the IOLs was counted under inversion phase microscope; Cell apoptosis test: after UVA illumination for different time(0 min、20 min、40min),cells were cultured for 5 hours, cell morphous was observed by TUNEL staining under ordinary light microscope and the apoptosis rate was evaluated by Image-Pro Plus analysis software. The surface energy components of the various substrates and HLECs were calculated based on Lifshitz-van del Waals/acid-base approach (LW-AB). The surface free energies obtained were used to calculate the interfacial free energies of adhesion (△Fadh ) of cells on various IOLs. Part Two: Platelets adhesion test:after the preparation of platelet rich plasma (PRP) by centrifugation, each IOL with the pre-irradiation for different minutes(0 min、10 min、30min) was cultured with 20μL PRP for 2 hours, then the platelets adhering to the IOL surface were observed with Wright staining and the adhesive rate was assessed by the analysis software; in Sterilization test:after the pre-irradiation of UVA for diffrernt time(0 min、10 min、30min), each IOL was placed under a culture of Staphylococcus aureus (S.A.,1×10~5 colony forming units [CFU]/mL) or Escherichia coli (E.C.BL21, 1×10~5 CFU /mL). After Gram staining, the survival percentage of S.A. was evaluated by software. The interfacial free energies of adhesion (△Fadh ) of two kinds of bacteria on various IOLs was calculated.
RESULTS: The first part of the study showed that as the time of ultraviolet irradiation increased, the contact angle of TiO_2/PMMA IOL grew down apparently(P﹤0.01) comparing with the control group. Cell adhesion test showed that as the pre-irradiation time increased, the number of the adhering cells was significantly lower in the TiO_2-coated IOL group than the noncoated IOL group (P﹤0.01). TUNEL staining showed that most HLECs began apoptosing when exposed to UVA for 20min. The TiO_2/PMMA IOL group had a higher apoptosis rate than PMMA IOL group(P﹤0.01). The second part: As the pre-irradiation time extended, the number of platelets and bacteria sticking on TiO_2/PMMA IOL reduced and the number was notably lesser in the TiO_2-coated IOL group than the noncoated IOL group (P﹤0.01). After UVA pre-irradiation for 30min, platelets and S.A. showed lower adhesive and survival rate on TiO_2/PMMA IOL surface. And Wright and Gram staining showed that both the distribution of platelets and bacteria on TiO_2/PMMA IOL suface were scattered singly, while most were clustered together on the PMMA IOL surface. The facial adhesion free energies of HLECs and bacteria to TiO_2/PMMA IOL were both positive(△Fadh﹥0), while PMMA IOL were negative. So TiO_2/PMMA IOLs would be more unfavourable for cells and bacteria adhesion from the point of view of thermodynamics.
CONCLUSION: (1) The TiO_2/PMMA IOLs coated with nano- TiO_2 thin film were shown to have smaller water contact angle and stronger hydrophilicity. And as the time of UVA irradiation extended, the angle got smaller and the hydrophilicity increased. (2) After UVA irradiation, the adhesion and growth of human lens epithelial cells, platelets, Staphylococcus aureus and Escherichia coli on the surface of TiO_2/PMMA IOL were all suppressed obviously. (3)The computation indicated that the facial adhesion free energy of HLECs, S. aureus and E. coli to TiO_2/PMMA IOLs elevated significantly, resulting an unfavourable intraocular surface for adhesion.
引文
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[10] Okajima Y, Saika S, Sawa M. Effects of surface modification on foreign body reaction of intraocular lenses[J]. Nippon Ganka Gakkai Zasshi, 2005,109 (5):267–273.
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[1] Trocme SD,Li H. Effect of heparin-surface-modified intraocular lenses on postoperative inflammation after Phacoemulsification:a randomized trial in a United States patient population[J].Ophthalmology,2000,107 (6):1031-1037.
[2] Tognetto D,Toto L,Ballone E,et a1.Biocompatibility of hydrophilic intraocular lenses[J].J Cataract Refract Surg ,2002,28 (4):644-651.
[3] Okajima Y, Saika S, Sawa M. Effects of surface modification on foreign body reaction of intraocular lenses[J]. Nippon Ganka Gakkai Zasshi, 2005,109 (5):267–273.
[4] Okajima Y, Saika S, Sawa M. Effect of surface coating an acrylic intraocular lens with poly(2-methacryloyloxyethyl phosphorylcholine) polymer on lens epithelial cell line behavior. J Cataract Refract Surg ,2006,32 (4):666-671.
[5] Shigeta M, Tanaka T, Koike N, et al. Suppression of fibroblast and bacterial adhesion by MPC coating on acrylic intraocular lenses[J]. J Cataract Refract Surg ,2006,32 (5):859–866.
[6] Yao K,Huang X-D,Huang X-J ,et al.Improvement of the surface biocompatibility of silicone intraocular lens by the plasma-induced tethering of phospholipid moieties[J].J Biomed Mater Res A ,2006,78 (4):684-692.
[7] 曲超,姚克,寇瑞强,等.а-烯丙基葡糖苷对聚甲基丙烯酸甲酯人工晶状体的表面修饰[J].生物医学工程学杂志,2004,21(1):115-117.
[8] 袁佳琴, 孙慧敏, 徐延山,等. 氟-肝素表面修饰人工晶状体的实验研究[J]. 眼科新进展, 2003,23(3):153-156.
[9] 王桂琴,彭秀军,顾汉卿.表面修饰硅凝胶人工晶状体植入兔眼的生物相容性[J]. 国际眼科杂志,2006,6(1):54-56.
[10]Yuan Z,Sun H,Yuan J . A 1-year study on carbon , titanium surface-modified intraocular lens in rabbit eyes[J].Graefes Arch Clin Exp Ophthalmol, 2004,242 (12):1008-1013.
[11] 黄晓丹,姚克,曲超,等.前表面磷脂修饰的硅凝胶人工晶状体表面生物学特性研究[J].中华眼科杂志,2007,43(2):167-169.
[12]Davidson MR, Mitchell SA, Bradley RH. UV-ozone modification of plasma-polymerised acetonitrile films for enhanced cell attachment[J]. Colloids Surf B Biointerfaces ,2004, 34 (4):213–219.
[13] Wang YX,Robertson JL,Spillman WB.Efects of the chemical structure and the surface properties of polymeric biomaterials on their biocompatibility[J].Pharm Res,2004,21(8):1362-1373.
[14] Matsushima H, Iwamoto H, Mukai K, et al.Active oxygen processing for acrylic intraocular lenses to prevent posterior capsule opacification[J].J Cataract Refract Surg, 2006,32(6):1035-1040.
[15] Yuen C, Williams R, Batterbury M, et al. Modification of the surface properties of a lens material to influence posterior capsular opacification[J]. Clin Experiment Ophthalmol, 2006,34 (6):568-574.
[16] Cooke C,McGimpsey S,Mahon G, et al.An in vitro study of human lens epithelial cell adhesion to intraocular lenses with and without a fibronectin coating[J].Invest Ophthalmol Vis Sci, 2006,47(7):2985-2989.
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