疏水性聚丙烯酸酯人工晶状体的大气压下辉光放电表面改性及其生物相容性研究
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摘要
第一部分大气压下辉光放电接枝聚乙二醇修饰疏水性聚丙烯酸酯人工晶状体及其体外生物相容性的研究
目的：
利用先进的大气压下辉光发电(APGD)技术接枝亲水性单体对人工晶状体(IOL)表面改性,探讨能够提高IOL生物相容性的简单、经济、高效的方法,以利于进一步的工业化生产。
方法：
采用APGD表面改性技术接枝亲水性单体聚乙二醇(PEG)修饰疏水性聚丙烯酸酯IOL前表面。应用静态水接触角(WCA)、X-射线光电子能谱(XPS)分别分析IOL表面亲水性和表面化学元素组成；场发射扫描电子显微镜(FESEM)和原子力显微镜(AFM)观察表面形貌；以考察改性后IOL表面理化性能的改变；按照医疗器械相关质量检测国家标准对IOL进行光焦度、像质、光谱透过率及动态疲劳耐久性的测试,以检测经PEG接枝后IOL的光学和力学性能。同时通过体外细胞粘附实验,包括血小板、巨噬细胞以及人晶状体上皮细胞(LECs),观察IOL表面所粘附细胞的数量、形态,以初步评价改性后IOL的体外生物相容性。
结果：
静态水接触角检测显示经APGD处理后的IOL前表面亲水性明显增强,又以PEG接枝后IOL为著。通过接枝不同分子量的PEG,筛选出α-PEGMA 1,100具有最佳改性效果,能长期保持亲水性,并应用于后续研究。XPS分析进一步证实了经APGD处理后IOL表面的极性基团的引入以及PEG的成功接枝。FESEM观察改性后IOL表面未见任何损伤；AFM显示APGD单纯处理导致表面粗糙度(RMS)增加,而在接枝PEG后得到改善。光学、力学相关检测证实IOL光焦度、像质、光谱透过率及动态抗疲劳强度均符合国家相关标准。体外血小板粘附实验显示APGD处理后的IOL可减少血小板粘附(p<0.05),接枝PEG后的IOL则该抑制效果更加明显(p<0.01),且粘附的血小板均处于球形的未被激活状态；巨噬细胞和LECs粘附结果也显示出类似的表现,APGD处理及PEG表面接枝均可抑制上述两类细胞的粘附,其中PEG接枝组还表现出明显的抑制LECs增殖的作用(p<0.01)。
结论：
APGD技术能够成功在疏水性聚丙烯酸酯IOL前表面接枝亲水性单体PEG,能够大大增强IOL的表面亲水性,减少表面细胞粘附,增加IOL前表面生物相容性。APGD表面改性技术操作流程简单、经济环保,可连续性作业,为实现进一步的工业化生产推广奠定基础。
第二部分疏水性聚丙烯酸酯人工晶状体经大气压下辉光放电接枝聚乙二醇修饰后的体内生物相容性研究
目的：
检测APGD前表面接枝PEG的疏水性聚丙烯酸酯IOL在动物眼内的生物相容性,即既能够改善与前表面相关的葡萄膜生物相容性,又能够保持疏水性聚丙烯酸酯IOL原有的良好囊膜相容性。
方法：
采用APGD表面改性方法在聚丙烯酸酯IOL前表面接枝PEG单体。静态接触角分析接枝后的IOL亲水性的稳定性。将36只比利时色素兔(36眼)分为三组,行标准超声乳化术后随机植入疏水性丙烯酸酯(Acrylic) IOL、PEG前表面接枝的IOL以及亲水性聚丙烯酸酯(Akreos) IOL,每一IOL组12只兔(12眼,右眼),术后常规用药。术后第1、3、7、14、30、60、90天,应用裂隙灯显微镜观察前房炎症反应、虹膜、瞳孔、IOL位置和囊膜等眼前节情况并按照相应评价标准进行评分,同时用后照法拍摄后囊膜照片经EPCO软件分析后囊膜混浊(PCO)情况。术后三月取材,Miyake-Apple后照法拍照进行PCO评分；取出IOL,行表面细胞Hematoxylin & Eosin staining (HE)染色细胞计数和扫描电子显微镜观察；眼球行石蜡包埋切片,HE染色、高碘酸-schiff (PAS)染色和三色法(Masson)染色后观察、晶状体囊膜、LECs细胞增殖情况及其与周围组织关系；免疫组织化学法观察collagenⅠ、collagenⅢ和α-SMA在囊膜及其周围的表达情况；透射电子显微镜超微结构分析晶状体囊膜中细胞与胞外基质的增殖状况。
结果：
接触角检测显示PEG接枝的IOL前表面为稳定的高度亲水性,而后表面保持疏水性不变。术后三个月观察期内,Acrylic IOL组的前房炎症反应较其他两组严重,有2眼发生虹膜后粘连和瞳孔夹持；Akreos IOL组有1眼发生瞳孔夹持、瞳孔变形；新型PEG接枝IOL组前房反应轻微。EPCO软件评分及Miyake-Apple后照法评价均显示Akreos IOL组的PCO评分显著高于PEG接枝IOL组和AcrylicIOL组,而后两者的PCO评分在统计学上无显著性差异。IOL表面细胞HE染色计数显示Acrylic IOL表面平均细胞级显著高于PEG修饰IOL和Akreos IOL。
组织病理、免疫组织化学以及透射电子显微镜结果均显示Acrylic IOL及PEG接枝IOL的后囊膜细胞纤维化趋势明显,与IOL贴附紧密,抑制了LECs向IOL光学区中央部的增殖移行,而Akreos IOL支持LECs及胞外基质在后囊膜及IOL后表面之间的增殖形成各类型的PCO。
结论：
应用APGD技术前表面接枝PEG的疏水性丙烯酸酯IOL植入兔眼内后,能减轻术后早期前房闪辉等炎症反应,降低IOL表面与炎性细胞的相互作用,并保持了疏水性聚丙烯酸酯IOL原有的低PCO发生率,兼具良好的葡萄膜相容性和囊膜相容性。进一步证实APGD表面接枝技术的体内安全性以及接枝的稳定性和有效性。
PartⅠModification of hydrophobic acrylic intraocular lens with poly(ethylene glycol) by atmospheric pressure glow discharge
Objective:
To improve the anterior surface biocompatibility of hydrophobic acrylic intraocular lens (IOL) in an efficient and continuous way, increase the anterior surface hydrophilicity while keeping the posterior surface hydrophobic, without change in the optical and mechanical properties of the bulk.
Methods:
Poly(ethylene glycol)s (PEGs) were immobilized by atmospheric pressure glow discharge (APGD) treatment using argon as the discharge gas. The hydrophilicity and chemical changes on the IOL surface were characterized by static water contact angle and X-ray photoelectron spectroscopy to confirm the covalent binding of PEG. The physic and optic properties were determined by national standards. The morphology of the IOL surface was observed under field emission scanning electron microscopy and atomic force microscopy. The surface biocompatibility was evaluated by adhesion experiments with platelets, macrophages and lens epithelial cells (LECs) in vitro.
Results:
The results revealed that the anterior surface of the PEG-grafted IOL displayed significantly and permanently improved hydrophilicity. The physic and optic properties of modified acrylic IOLs meet the national standards (including optical power, image quality, spectral transmittance and dynamic fatigue durability). Cell repellency was observed, especially in the PEG-modified IOL group, which resisted the attachment of platelets, macrophages and LECs. Moreover, the spread and growth of cells were suppressed, and may be attributed to the steric stabilization force and chain mobility effect of the modified PEG.
Conclusion:
All of these results indicated that hydrophobic acrylic IOLs can be hydrophilic modified by PEG through APGD treatment in a convenient and continuous manner and will provide advantages for further industrial applications.
Part II The biocompatibility of anterior surface PEG-modified acrylic IOLs prepared via APGD treatment in vivo
Objective:
To detect the biocompatibility of anterior surface PEG-grafted IOLs (via APGD treatment) in rabbit eyes, including complications related to anterior chamber inflammatory reaction and capsule opacification.
Methods:
Anterior surfaces of hydrophobic acrylic IOLs were grafted with PEG by APGD treatment. The hydrophilicity of IOL surface was characterized by contact angle test. Thirty-six rabbit eyes were operated on with phacoemulsification and randomly implantation of one from the three types of foldable IOLs:anterior surface PEG-grafted acrylic IOL (PEG-grafted IOL) (n=12), original acrylic IOL (Acrylic IOL) (n=12) and hydrophilic IOL (Akreos IOL) (n=12). Postoperative follow-up was done on the 1st,3rd, 7th,14th,30th,60th and 90th days after surgery. On each visit, all rabbits were observed by slitlamp examination to evaluate aqueous flare, aqueous cell, posterior synechia and IOL dislocation. Standardized digital retroilluminated slitlamp images of posterior capsule opacification (PCO) were taken and the evaluation of PCO was done by EPCO 2000 software. The rabbits were killed three months postoperatively, the Miyake-Apple posterior photographic technique was used to evaluate the PCO formation. The IOLs were extracted from the rabbits eyes, and the cells attached on IOLs surface were observed by inverted phase contrast microscopy with hematoxylin and eosin (HE) staining; the samples were also prepared and observed by SEM. Histological sections of rabbits globe were prepared and stained with HE staining, periodic acid-schiff (PAS) and Masson's trichrome staining to document proliferation of LECs and extracellular matrix (ECM) in capsular bag. Immunohistochemistry (IHC) for collagenⅠ, collagenⅢandα-SMA was also done to evaluate mesenchymal transition in LECs. Both the anterior and posterior capsules were processed for transmission electron microscopy (TEM) to observe the microstructure of lens capsule.
Results:
The water contact angle of PEG IOL showed a stable significantly improved hydrophilicity of anterior surface, with a hydrophobic original posterior surface. The follow up observation with slitlamp examination showed that the anterior chamber inflammation was severer in Acrylic IOL group than in other two groups, two eyes in Acrylic IOL group and one eye in Akreos IOL group presented posterior synechia and IOL dislocation, no obvious inflammation was found in eyes with PEG IOLs. On the 7th and 14th day postoperatively, the aqueous flare results were statistically higher in Acrylic IOL group than in other two groups (p<0.05), the aqueous cell results showed that no statistically difference among the three groups was observed in the measured time point (p>0.05). The EPCO analysis and Miyake-Apple evaluation revealed that the eyes of Akreos IOLs had higher incidence of PCO compared to those of PEG IOLs and Acrylic IOLs (p<0.05), but there were no statistically significant difference between PEG IOLs and Acrylic IOL groups (p>0.05). The inverted phase contrast microscope observation showed that there were more cells adhered on Acrylic IOLs than PEG IOLs and Akreos IOLs (p<0.05). Histopathological, IHC and TEM analyses demonstrated the remnant LECs had some growth, and Soemmering's ring was observed in all groups. However, LECs were stopped at the optic periphery of the Acrylic IOLs and PEG IOLs, which keeping hydrophobic posterior surface. The central posterior capsule area of IOLs were clear in above both groups. In contrast, in Akreos IOL group, the LECs and ECM were observed apparently grew into the central field of the optic, thus leading to the PCO in visual axis area.
Conclusion:
The PEG IOLs via APGD treatment had stable hydrophilic anterior surface and original hydrophobic posterior surface. The PEG IOLs have both excellent uveal biocompatibility, characterized by minimal postoperative anterior chamber inflammation, and good capsular biocompatibility, characterized by low incidence of PCO.
目的：
利用先进的大气压下辉光发电(APGD)技术接枝亲水性单体对人工晶状体(IOL)表面改性,探讨能够提高IOL生物相容性的简单、经济、高效的方法,以利于进一步的工业化生产。
方法：
采用APGD表面改性技术接枝亲水性单体聚乙二醇(PEG)修饰疏水性聚丙烯酸酯IOL前表面。应用静态水接触角(WCA)、X-射线光电子能谱(XPS)分别分析IOL表面亲水性和表面化学元素组成；场发射扫描电子显微镜(FESEM)和原子力显微镜(AFM)观察表面形貌；以考察改性后IOL表面理化性能的改变；按照医疗器械相关质量检测国家标准对IOL进行光焦度、像质、光谱透过率及动态疲劳耐久性的测试,以检测经PEG接枝后IOL的光学和力学性能。同时通过体外细胞粘附实验,包括血小板、巨噬细胞以及人晶状体上皮细胞(LECs),观察IOL表面所粘附细胞的数量、形态,以初步评价改性后IOL的体外生物相容性。
结果：
静态水接触角检测显示经APGD处理后的IOL前表面亲水性明显增强,又以PEG接枝后IOL为著。通过接枝不同分子量的PEG,筛选出α-PEGMA 1,100具有最佳改性效果,能长期保持亲水性,并应用于后续研究。XPS分析进一步证实了经APGD处理后IOL表面的极性基团的引入以及PEG的成功接枝。FESEM观察改性后IOL表面未见任何损伤；AFM显示APGD单纯处理导致表面粗糙度(RMS)增加,而在接枝PEG后得到改善。光学、力学相关检测证实IOL光焦度、像质、光谱透过率及动态抗疲劳强度均符合国家相关标准。体外血小板粘附实验显示APGD处理后的IOL可减少血小板粘附(p<0.05),接枝PEG后的IOL则该抑制效果更加明显(p<0.01),且粘附的血小板均处于球形的未被激活状态；巨噬细胞和LECs粘附结果也显示出类似的表现,APGD处理及PEG表面接枝均可抑制上述两类细胞的粘附,其中PEG接枝组还表现出明显的抑制LECs增殖的作用(p<0.01)。
结论：
APGD技术能够成功在疏水性聚丙烯酸酯IOL前表面接枝亲水性单体PEG,能够大大增强IOL的表面亲水性,减少表面细胞粘附,增加IOL前表面生物相容性。APGD表面改性技术操作流程简单、经济环保,可连续性作业,为实现进一步的工业化生产推广奠定基础。
第二部分疏水性聚丙烯酸酯人工晶状体经大气压下辉光放电接枝聚乙二醇修饰后的体内生物相容性研究
目的：
检测APGD前表面接枝PEG的疏水性聚丙烯酸酯IOL在动物眼内的生物相容性,即既能够改善与前表面相关的葡萄膜生物相容性,又能够保持疏水性聚丙烯酸酯IOL原有的良好囊膜相容性。
方法：
采用APGD表面改性方法在聚丙烯酸酯IOL前表面接枝PEG单体。静态接触角分析接枝后的IOL亲水性的稳定性。将36只比利时色素兔(36眼)分为三组,行标准超声乳化术后随机植入疏水性丙烯酸酯(Acrylic) IOL、PEG前表面接枝的IOL以及亲水性聚丙烯酸酯(Akreos) IOL,每一IOL组12只兔(12眼,右眼),术后常规用药。术后第1、3、7、14、30、60、90天,应用裂隙灯显微镜观察前房炎症反应、虹膜、瞳孔、IOL位置和囊膜等眼前节情况并按照相应评价标准进行评分,同时用后照法拍摄后囊膜照片经EPCO软件分析后囊膜混浊(PCO)情况。术后三月取材,Miyake-Apple后照法拍照进行PCO评分；取出IOL,行表面细胞Hematoxylin & Eosin staining (HE)染色细胞计数和扫描电子显微镜观察；眼球行石蜡包埋切片,HE染色、高碘酸-schiff (PAS)染色和三色法(Masson)染色后观察、晶状体囊膜、LECs细胞增殖情况及其与周围组织关系；免疫组织化学法观察collagenⅠ、collagenⅢ和α-SMA在囊膜及其周围的表达情况；透射电子显微镜超微结构分析晶状体囊膜中细胞与胞外基质的增殖状况。
结果：
接触角检测显示PEG接枝的IOL前表面为稳定的高度亲水性,而后表面保持疏水性不变。术后三个月观察期内,Acrylic IOL组的前房炎症反应较其他两组严重,有2眼发生虹膜后粘连和瞳孔夹持；Akreos IOL组有1眼发生瞳孔夹持、瞳孔变形；新型PEG接枝IOL组前房反应轻微。EPCO软件评分及Miyake-Apple后照法评价均显示Akreos IOL组的PCO评分显著高于PEG接枝IOL组和AcrylicIOL组,而后两者的PCO评分在统计学上无显著性差异。IOL表面细胞HE染色计数显示Acrylic IOL表面平均细胞级显著高于PEG修饰IOL和Akreos IOL。
组织病理、免疫组织化学以及透射电子显微镜结果均显示Acrylic IOL及PEG接枝IOL的后囊膜细胞纤维化趋势明显,与IOL贴附紧密,抑制了LECs向IOL光学区中央部的增殖移行,而Akreos IOL支持LECs及胞外基质在后囊膜及IOL后表面之间的增殖形成各类型的PCO。
结论：
应用APGD技术前表面接枝PEG的疏水性丙烯酸酯IOL植入兔眼内后,能减轻术后早期前房闪辉等炎症反应,降低IOL表面与炎性细胞的相互作用,并保持了疏水性聚丙烯酸酯IOL原有的低PCO发生率,兼具良好的葡萄膜相容性和囊膜相容性。进一步证实APGD表面接枝技术的体内安全性以及接枝的稳定性和有效性。
PartⅠModification of hydrophobic acrylic intraocular lens with poly(ethylene glycol) by atmospheric pressure glow discharge
Objective:
To improve the anterior surface biocompatibility of hydrophobic acrylic intraocular lens (IOL) in an efficient and continuous way, increase the anterior surface hydrophilicity while keeping the posterior surface hydrophobic, without change in the optical and mechanical properties of the bulk.
Methods:
Poly(ethylene glycol)s (PEGs) were immobilized by atmospheric pressure glow discharge (APGD) treatment using argon as the discharge gas. The hydrophilicity and chemical changes on the IOL surface were characterized by static water contact angle and X-ray photoelectron spectroscopy to confirm the covalent binding of PEG. The physic and optic properties were determined by national standards. The morphology of the IOL surface was observed under field emission scanning electron microscopy and atomic force microscopy. The surface biocompatibility was evaluated by adhesion experiments with platelets, macrophages and lens epithelial cells (LECs) in vitro.
Results:
The results revealed that the anterior surface of the PEG-grafted IOL displayed significantly and permanently improved hydrophilicity. The physic and optic properties of modified acrylic IOLs meet the national standards (including optical power, image quality, spectral transmittance and dynamic fatigue durability). Cell repellency was observed, especially in the PEG-modified IOL group, which resisted the attachment of platelets, macrophages and LECs. Moreover, the spread and growth of cells were suppressed, and may be attributed to the steric stabilization force and chain mobility effect of the modified PEG.
Conclusion:
All of these results indicated that hydrophobic acrylic IOLs can be hydrophilic modified by PEG through APGD treatment in a convenient and continuous manner and will provide advantages for further industrial applications.
Part II The biocompatibility of anterior surface PEG-modified acrylic IOLs prepared via APGD treatment in vivo
Objective:
To detect the biocompatibility of anterior surface PEG-grafted IOLs (via APGD treatment) in rabbit eyes, including complications related to anterior chamber inflammatory reaction and capsule opacification.
Methods:
Anterior surfaces of hydrophobic acrylic IOLs were grafted with PEG by APGD treatment. The hydrophilicity of IOL surface was characterized by contact angle test. Thirty-six rabbit eyes were operated on with phacoemulsification and randomly implantation of one from the three types of foldable IOLs:anterior surface PEG-grafted acrylic IOL (PEG-grafted IOL) (n=12), original acrylic IOL (Acrylic IOL) (n=12) and hydrophilic IOL (Akreos IOL) (n=12). Postoperative follow-up was done on the 1st,3rd, 7th,14th,30th,60th and 90th days after surgery. On each visit, all rabbits were observed by slitlamp examination to evaluate aqueous flare, aqueous cell, posterior synechia and IOL dislocation. Standardized digital retroilluminated slitlamp images of posterior capsule opacification (PCO) were taken and the evaluation of PCO was done by EPCO 2000 software. The rabbits were killed three months postoperatively, the Miyake-Apple posterior photographic technique was used to evaluate the PCO formation. The IOLs were extracted from the rabbits eyes, and the cells attached on IOLs surface were observed by inverted phase contrast microscopy with hematoxylin and eosin (HE) staining; the samples were also prepared and observed by SEM. Histological sections of rabbits globe were prepared and stained with HE staining, periodic acid-schiff (PAS) and Masson's trichrome staining to document proliferation of LECs and extracellular matrix (ECM) in capsular bag. Immunohistochemistry (IHC) for collagenⅠ, collagenⅢandα-SMA was also done to evaluate mesenchymal transition in LECs. Both the anterior and posterior capsules were processed for transmission electron microscopy (TEM) to observe the microstructure of lens capsule.
Results:
The water contact angle of PEG IOL showed a stable significantly improved hydrophilicity of anterior surface, with a hydrophobic original posterior surface. The follow up observation with slitlamp examination showed that the anterior chamber inflammation was severer in Acrylic IOL group than in other two groups, two eyes in Acrylic IOL group and one eye in Akreos IOL group presented posterior synechia and IOL dislocation, no obvious inflammation was found in eyes with PEG IOLs. On the 7th and 14th day postoperatively, the aqueous flare results were statistically higher in Acrylic IOL group than in other two groups (p<0.05), the aqueous cell results showed that no statistically difference among the three groups was observed in the measured time point (p>0.05). The EPCO analysis and Miyake-Apple evaluation revealed that the eyes of Akreos IOLs had higher incidence of PCO compared to those of PEG IOLs and Acrylic IOLs (p<0.05), but there were no statistically significant difference between PEG IOLs and Acrylic IOL groups (p>0.05). The inverted phase contrast microscope observation showed that there were more cells adhered on Acrylic IOLs than PEG IOLs and Akreos IOLs (p<0.05). Histopathological, IHC and TEM analyses demonstrated the remnant LECs had some growth, and Soemmering's ring was observed in all groups. However, LECs were stopped at the optic periphery of the Acrylic IOLs and PEG IOLs, which keeping hydrophobic posterior surface. The central posterior capsule area of IOLs were clear in above both groups. In contrast, in Akreos IOL group, the LECs and ECM were observed apparently grew into the central field of the optic, thus leading to the PCO in visual axis area.
Conclusion:
The PEG IOLs via APGD treatment had stable hydrophilic anterior surface and original hydrophobic posterior surface. The PEG IOLs have both excellent uveal biocompatibility, characterized by minimal postoperative anterior chamber inflammation, and good capsular biocompatibility, characterized by low incidence of PCO.
引文
[1]赵家良.我国防盲治盲工作的进展.中华眼科杂志2005；41(8)：697-701.
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