折叠式人工玻璃体填充聚乙烯醇水凝胶重建自然玻璃体的实验研究
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摘要
背景
玻璃体是一种透明的不可再生的凝胶体,其主要由99%的水与1%的无机盐、胶原和透明质酸等构成,胶原纤维呈三维网状结构,其上附着透明质酸粘多糖,后者能与水分子结合,从而使玻璃体呈凝胶状。玻璃体的主要生理功能是支撑视网膜、眼屈光、细胞屏障和营养等。由于自然玻璃体的不可再生性,因此,当发生玻璃体视网膜疾病时,如：外伤导致的视网膜脱离、外伤性增生性玻璃体视网膜病变(T-PVR)、增生性玻璃体视网膜病变(PVR)、增生性糖尿病视网膜病变(PDR)、严重眼内炎等等,需行玻璃体切割手术治疗,术中切除自然玻璃体后必须填充合适的人工玻璃体替代物,修复眼损伤,支撑视网膜,重建视功能,防止眼球萎缩。
目前,研究者们对玻璃体替代物的研究甚多,临床常用的主要有空气、惰性气体、重水、硅油、重硅油等。使用的方式均为直接注入至玻璃体腔,与视网膜直接接触,利用其表面张力而达到支撑视网膜的效果。以上除硅油外,均为短期应用。然而,长期的硅油填充,容易导致一系列的术后并发症,如并发性白内障、继发性青光眼、角膜变性、硅油乳化、甚至可移行至视网膜下和视神经,造成视神经纤维的髓鞘脱失,导致视力永久丧失。因此,颇为亟待寻找一种具有良好生物相容性,无严重并发症,并能长期填充的人工玻璃体。
水凝胶因具有良好的生物相容性、光学性能、减震性,能模拟自然玻璃体的特性,而被誉为是人工玻璃体最好的候选者,从上世纪九十年代以来一直是国内外的研究热点。主要有聚乙烯醇[poly(vinyl alcohol), PVA]水凝胶、聚乙二醇[polyethylene glycol), PEG]水凝胶、聚丙烯酸[poly(acrylic acid), PAA]水凝胶和聚丙烯酰胺[polyacrylamide, PAM]水凝胶等。研究者们可体外通过改变水凝胶的合成过程,来满足自然玻璃体的物理性能,然后将其注入体内。也可以将液态的高分子物质注射入玻璃体腔后进行原位交联而形成水凝胶,这就可避免注射过程中对水凝胶结构的破坏。同时,水凝胶还可以负载药物,作为眼内药物释放的载体。作为短期的玻璃体替代物,水凝胶在动物实验眼中应用并没有明显的术后并发症。但由于其注入玻璃体腔后,与视网膜直接接触,使得水凝胶参与眼内代谢和循环,导致在眼内容易被降解和吸收。因此,仍然不能作为长期的玻璃体替代物,不能满足对治疗严重的视网膜脱离等疾病需要长期顶压的要求。所以,如何减少或阻止水凝胶在眼内的降解吸收,延长水凝胶在玻璃体腔的停留时间,使之能成为一种长期填充的人工玻璃体是眼科研究一个非常重要的科学问题。
自然玻璃体有一层薄膜包围,此薄膜属于解剖学玻璃体皮质范畴。由于年龄、外伤和视网膜玻璃体病变等因素,薄膜结构可遭到破坏,受此启发,我们研究总结发现目前人工玻璃体的研究方法是直接注射,都没有解决人工玻璃体和眼内组织直接接触导致的各种问题。需要研究一个能像自然玻璃体一样的囊膜包绕人工玻璃体,限制人工玻璃体的眼内流动,避免或减少被吸收和变质,从而延长人工玻璃体的使用时间。
因此,我们首次提出一个替代自然玻璃体的新策略-折叠式人工玻璃体(FCVB),它主要由球囊、引流管和引流阀组成。球囊部是通过计算机精细模拟人眼玻璃体制作而成,并且通过引流管与引流阀相连接,由能在体内应用20年以上的硅橡胶薄膜制成。通过微创切口将球囊部植入到玻璃体腔,随后注入流动介质,如生理盐水、硅油、水凝胶等,形成玻璃体形状的薄膜球囊支撑视网膜、充填眼球。
折叠式人工玻璃体避免了目前人工玻璃体的严重缺点,可以精细模拟自然玻璃体的结构,恢复自然玻璃体的视网膜支撑、眼屈光、细胞屏障等主要生理功能。并通过从动物实验、标准制定到临床试验的系列研究证实,折叠式人工玻璃体填充硅油在眼内长期填充后显示出良好的生物相容性,有效的360。全方位支撑视网膜,促进脱离视网膜的复位,能显著减少硅油诱导的并发症如硅油乳化、继发性青光眼、角膜变性等。但是,由于折叠式人工玻璃体填充硅油后无法恢复自然玻璃体的屈光性能,没有恢复自然玻璃体所拥有的机械减震和粘弹性能。
目的
因此,结合水凝胶的国际研究进展,本课题提出应用折叠式人工玻璃体填充聚乙烯醇(PVA)水凝胶重建自然玻璃体的设想。利用辐照法交联得到不同浓度的PVA水凝胶,体外检测光学、物理、机械性能及细胞毒性等,筛选最接近于人自然玻璃体性能的PVA水凝胶；在兔眼模型中全面评估FCVB填充PVA水凝胶作为玻璃体替代物的光学物理性能、视网膜支撑功能和生物相容性等。本研究旨在探讨FCVB填充PVA水凝胶作为玻璃体替代物,具有良好的生物相容性,能够避免PVA水凝胶与视网膜的直接接触,阻止PVA水凝胶参与眼内代谢和循环,减少PVA水凝胶的降解和吸收,延长水凝胶在玻璃体腔内的停留时间,恢复自然玻璃体的屈光、视网膜支撑和粘弹性能,从而达到重建自然玻璃体的目的。
材料和方法
第一部分：聚乙烯醇(PVA)水凝胶的制备及其性能与生物相容性研究
1.PVA水凝胶的制备：聚乙烯醇(PVA)购于Sigma Aldrich公司。配制不同浓度(1%、3%、7%,质量-体积分数)PVA溶液,采用γ-射线(Co60)辐照交联得到1%PVA水凝胶、3%PVA水凝胶、7%PVA水凝胶。将水凝胶分别浸入双蒸水中浸泡48h,以除去未交联PVA单体。
2.物理与光学性能：测量充分溶胀后的PVA水凝胶的比重、含水率、透光率、屈光率、pH值、溶胀性能等参数。
3.流变性能：PVA水凝胶经充分溶胀后,应用ARES-RFS流变仪对水凝胶行动态应变扫描测试、粘弹性能和蠕变性能测试。测量温度为37±0.1℃。动态应变扫描测试：在振荡频率1Hz条件下,确定线性粘弹区间的应变振幅后；在该应变振幅下,检测水凝胶在振荡频率范围为0.01-10Hz时储存模量(G')和损耗模量(G")的动态变化。蠕变测试,在恒定剪切应力σ0=0.1Pa条件下,检测水凝胶随着剪切应力时间的延长(500s),其应变(力反应的变化趋势。用同样方法测量硅油和透明质酸酸钠的流变性能。通过对比选取其中一种与自然玻璃体参数最为相似的PVA水凝胶。
4.体外细胞毒性试验：采用噻唑蓝比色法(MTT法)测定PVA水凝胶对L929鼠成纤维细胞生长抑制作用。制备待测样本浸泡液：样本分为6组(1%PVA、3%PVA、7%PVA、FCVB+1%PVA、FCVB+3%PVA、FCVB+7%PVA),分别将样本放入DMEM培养基浸泡24h、48h、72h,收集浸泡液备用。取对数生长期细胞,接种于96孔培养板,每孔加100μL细胞悬液(每孔5000个细胞)细胞贴壁后,加入不同样本的浸泡液,以培养液作为对照。将培养板置入37℃、5%C02培养箱中,分别培养24h、48h、72h。显微镜下观察细胞形态和行MTT比色检测,酶标仪于波长570nm下测定OD值并计算抑制率,评价样品的细胞毒性。
5.统计学分析：采用SPSS13.0统计软件进行分析,所有计量资料用均数±标准差(X±SD)表示。MTT比色试验中,不同时间点及不同组OD值用单个重复测量因素方差分析,P<0.05认为具有统计学显著差异。
第二部分：折叠式人工玻璃体(FCVB)填充PVA水凝胶重建自然玻璃体的体内研究
1.动物手术：选取体重2.5-3kg新西兰白兔18只,所有实验操作严格遵守ARVO关于实验动物使用的规定。分为三组：PVA水凝胶组(PVA组)、FCVB+PVA水凝胶组(FCVB+PVA组)、平衡盐液组(BSS组)。右眼行常规三通道玻璃体切割术,切除玻璃体,术中填充PVA水凝胶6眼、填充FCVB+PVA水凝胶6眼、填充BSS溶液6眼。术后常规予典必殊滴眼液、普南扑灵滴眼液和百力特滴眼液滴术眼抗炎预防感染治疗。
2.术后观察：术后3、7、14、30、60、90、180天,常规行裂隙灯、直接眼底镜、眼压和眼底照相检查,观察术眼前节和眼底情况；术后14、30、60、90和180天行术眼B超检查了解玻璃体腔和眼底视网膜解剖位置情况；术后90、180天,分别行双眼ERG检查,评估玻璃体替代物植入后对视网膜功能的影响。
3.眼球病理检测：于术后90天(PVA组,3只；FCVB+PVA组,3只；BSS组,3只)和180天(PVA组,3只；FCVB+PVA组,3只；BSS组,3只),用过量利多卡因麻醉处死兔子,剜出眼球,马上取出并收集眼内PVA水凝胶后,迅速将眼球迅速放入固定液中,固定眼球,按常规方法做石蜡切片光学显微镜、HE染色检查。
4.PVA水凝胶性能检测：将眼内填充后的PVA水凝胶分别行透光率、屈光率和流变学检查。方法同前。
5.统计学分析：采用SPSS13.0统计软件进行分析,所有计量资料用均数±标准差(X±SD)表示。眼压用单个重复测量因素方差分析,ERG振幅值用单因素方差分析(One-way ANOVA),方差齐时采用S-N-K法进行多重比较,方差不齐时采用近似F检验Welch法进行校正,采用Dunnett T3法进行多重比较。P<0.05认为具有统计学显著差异。
结果
第一部分：聚乙烯醇(PVA)水凝胶的制备及其性能与生物相容性研究
1.光学及物理性能：1%PVA水凝胶的含水率为98.9%,屈光指数为1.3355,透光率为94.8%,pH值为7.22,比重为1.0039；3%PVA水凝胶的含水率为98.1%,屈光指数为1.3361,透光率为93.2%,pH值为7.25,比重为1.0144；7%PVA水凝胶的含水率为93.5%,屈光指数为1.3425,透光率为88%,pH值为7.41,比重为1.1147；自然玻璃体的含水率为98-99%,屈光指数为1.3345-1.3348,透光率为>90%,pH值为7.0-7.4,比重为1.0053-1.0089。
2.流变性能：三种浓度PVA水凝胶储存模量(G’)均大于损耗模量(G"),表明三种物质均为有粘弹性能的凝胶体,且其弹性性能大于粘性性能。而硅油表现为损耗模量(G")大于储存模量(G'),提示其粘性性能大于弹性性能,因此主要表现为粘性液体的状态。1%PVA:G'为3.2±1.1Pa,G"为0.8±0.5Pa；3%PVA:G'为6.1±1.3Pa,G"为1.3±0.9Pa:7%PVA:G'为106.5±18.6Pa,G''为18.3±12.8Pa；硅油：G'为2.4±1.6Pa,G"为44.8±28.3Pa;透明质酸：G'为155.2±147.2Pa,G"为92.9±49.1Pa。自然玻璃体：G'为2.8±0.9Pa,G"为0.7±0.4Pa
在顺应性方面,良好的顺应性使得在玻璃体腔内可以拥有更为良好的机械性能,7%PVA>3%PVA>1%PVA;在蠕变方面,它代表的是迅时弹性形变的能力和柔韧性,3%PVA>7%PVA>1%PVA。综合以上光学、物理和流变性能,可以得出3%PVA水凝胶的性能更为接近自然玻璃体,因此选择3%PVA水凝胶作为FCVB填充物进行体内的进一步研究。
3.细胞毒性：6组(1%PVA、3%PVA、7%PA、FCVB+1%PVA、 FCVB+3%PVA、FCVB+7%PVA)材料样品浸泡提取液与L929细胞共培养24、48及72h后,光镜下观察细胞形态和结构均无显著变化。MTT比色法测定OD值,提示在24、48、72h三个时间点,1%PVA、3%PVA、7%PVA组及FCVB+1%PvA、FCVB+3%PVA、FCVB+7%PVA组与对照组,OD值比较均无显著统计学差异(P>0.05)。所以,结果提示FCVB填充PVA水凝胶后或单纯PVA水凝胶均无明显细胞毒性。
第二部分：折叠式人工玻璃体(FCVB)填充PVA水凝胶重建自然玻璃体的体内研究
1.裂隙灯：术后3天,PVA组及BSS组均出现了不同程度结膜水肿、充血和轻度前房炎症反应,至术后7天症状全部消失；而FCVB+PVA组在术后7天前房仍出现少量炎性细胞,但未见瞳孔后粘连及纤维素样渗出,给予积极抗炎防感染治疗后,到术后14天结膜水肿、充血及前房炎症逐渐消退。在随后长达半年的随访观察,三组白兔术眼的眼前节均为出现明显异常,但是PVA组和BSS组有术眼出现不同程度的晶体混浊,术后90天时白内障发生率：PVA组为16.7%(1/6)；术后180天时白内障发生率：PVA组为33.3%(1/3),BSS组为16.7%(1/6)；由于先前研究发现兔眼植入FCVB后白内障的发生率较高,考虑本研究观察时间较长,白内障严重后会影响玻璃体腔和眼底视网膜等组织的观察,为避免二次手术,本研究中FCVB+PVA组采取行玻璃体切割手术的同时行晶体切除术。
2.眼底检查：术后14天,FCVB+PVA组随着炎症不断消退,眼底可隐约见视盘和血管,至术后30天,眼底能清晰可见,直至观察终点术后180天,玻璃体腔仍然保持透明,能清晰看到眼底视网膜、血管、视盘等结构。在PVA组,术后14天时能清晰看到眼底,然而随着白内障的发展和进一步加重,术后90天和180天,眼底逐渐变得模糊,但是仍可见眼底视盘、血管、视网膜等大体结构。BSS组在180天的观察期内,眼底均可清晰可见。随访观察期内三组术眼均未见有玻璃体出血、混浊、玻璃体视网膜增殖和视网膜脱离等情况发生。
3.眼压：术后第3天,FCVB+PVA组眼压呈现下降趋势,与术前比较有显著统计学差异(P<0.05),但到术后7天,眼压逐渐上升至正常范围,与术前比较无显著统计学差异(P>0.05),至术后14天、30天、60天、90天、180天,眼压一直维持在正常范围,与术前比较无显著统计学差异(P>0.05)。在PVA组和BSS组,术后3天眼压出现明显的升高,与术前相比有显著统计学差异(P<0.05),但到术后7天,眼压逐渐降低至正常范围,与术前比较无显著统计学差异(P>0.05),至术后14天、30天、60天、90天,眼压维持较平稳,与术前比较无显著统计学差异(P>0.05)。但是在术后180天,PVA组眼压与术前相比呈显著下降趋势(P<0.05)。
4.眼部B超：在术后整个180天的随访观察期内,三组兔眼眼部B超均未显示有玻璃体混浊、玻璃体腔出血、增殖或视网膜脱离、脉络膜脱离等声像。FCVB+PVA组眼底B超提示FCVB囊壁与视网膜相贴,良好的支撑视网膜。
5.眼部电生理：全视网膜电图(ERG)检查提示：FCVB+PVA组,术后90天和术后180天,a波和b波峰值均比术前下降,差异具有统计学意义(P<0.05)。在PVA组和BSS组,术后90和180天a波和b波峰值与术前变化比较差异无统计学意义(P>0.05)。
6.眼球病理：FCVB+PVA组,术后90、180天剜出眼球,大体解剖观察可见眼球充实饱满,FCVB填充PVA水凝胶后能良好的支撑视网膜,取出FCVB球囊内的PVA水凝胶仍然透明和维持较好的粘弹性。PVA组,术后90天摘出眼球饱满充实,取出眼内PVA水凝胶亦保持透明和一定的粘弹性；但是在术后180天时,摘出的眼球外观轻度萎缩,取出眼内PVA水凝胶虽然透明,但是明显被降解和吸收,间中混有较多的水样物质,粘弹性明显减弱。取出眼球固定后,石蜡固定切片,HE染色,提示：除FCVB+PVA组在术后180天视网膜出现结构紊乱、神经纤维层变薄,神经节细胞结构破坏等情况外,其余均未见明显结构异常包括角膜、睫状体、视网膜等。
7.光学性能与流变学检查：术后90天,FCVB+PVA组与PVA组填充后的PVA水凝胶其光学和流变性能和填充前相比无显著改变。在FCVB+PVA组,PVA水凝胶填充180天后,其光学与流变性能和填充前相比仍然无明显改变,PVA水凝胶无明显降解和吸收。然而在PVA组,PVA水凝胶填充180天后,其折光率和流变性能较前发生明显改变,折光率降低为1.3121,储存模量(G')从6.1±1.3降至3.9±0.8Pa,损耗模量(G")从1.3±0.9降至0.8±0.5Pa,粘弹性能明显降低,提示经180天眼内填充后PVA水凝胶显著被降解和吸收。
结论
1.3%PVA水凝胶具有与自然玻璃体相似的光学、物理和流变性能,拥有良好的生物相容性,能很好的模拟自然玻璃体的性能。
2. FCVB填充3%PVA水凝胶作为一个新的玻璃体替代物,经兔眼玻璃体腔内的长期(180天)填充后,发现其具有良好的光学性能、机械性能和生物相容性,能够安全有效、全方位的支撑视网膜,维持视网膜的解剖结构；FCVB能有效的发挥生物屏障的功能,阻止PVA水凝胶的快速降解和吸收,延长PVA水凝胶在玻璃体内的填充时间,为寻找一种理想的玻璃体替代物提供新的思路和方法。
Background
The vitreous body is a clear, non-renewable gel body, which is mainly constituted by the99%water and1%inorganic salts, collagen and hyaluronic acid. Collagen fibers are three-dimensional mesh structure, attached by hyaluronic mucopolysaccharide, which can be combined with water molecules, so that the vitreous is substantially gelled. The physiological functions of vitreous body are supporting the retina, refraction, cell barriers and nutrition in eyes. Due to the non-renewable properties of natural vitreous body, when vitreoretinal diseases occur, such as:the trauma caused retinal detachment, traumatic proliferative vitreoretinopathy (T-PVR), proliferative vitreoretinopathy (PVR), proliferative diabetic retinopathy (PDR) and so on, vitrectomy surgery is needed. After intraoperative resection of natural vitreous body, suitable artificial vitreous substitutes must be filled into vitreous cavities to repair the damage, supporting retina, reconstructing visual function to prevent atrophy of eyeball.
Currently, the vitreous substitutes commonly used in clinical include air, inert gas, heavy water, silicone oil, heavy silicone oil and so on. All of them are directly injected into the vitreous cavity, directly contacting with the retina, to support retina by the surface tension. The others are short-term tamponade in vivo in addition to silicone oil. However, after a long-term tamponade in vitreous cavity, a series of post-operative complications are likely to occur, such as cataract, secondary glaucoma, corneal degeneration, silicone oil emulsification and even migrate to subretina or optic nerve, causing demyelination of the optic nerve fibers, resulting in permanent loss of vision. Therefore, it is quite urgent to find an ideal vitreous substitute with good biocompatibility without serious complications for long-term tamponade.
Hydrogel is known as one of the best artificial vitreous candidates because of its good biocompatibility, optical property and shock absorption performances. It can mimic the characteristics of natural vitreous, and has been a hot research focus at home and abroad since the1990s. Hydrogel mainly includes PVA [poly (vinyl alcohol)] hydrogel, PEG [poly (ethylene glycol)] hydrogel, PAA [poly (acrylic acid)] hydrogel, PAM [poly acrylamide] hydrogel, and so on. To meet the physical properties of natural vitreous, researchers have been trying to change different synthesis process of the hydrogel in vitro, and then injected into the vitreous cavity. Liquid polymeric substance may also be injected into the vitreous cavity which is crosslinked in situ to form hydrogel in vitreous cavity. In this way, it can avoid the structure destruction during injection of the hydrogel. Additionally, the hydrogel can load drugs, acting as a release vector of drug within the eye. There were no significant postoperative complications after a short-term tamponade in animal experiments. Because all the substitutes are directly injected into the vitreous cavity, directly contacting with the retina, the hydrogels are also easily involved in the metabolism and circulation within the eye, resulting in rapidly degradation and absorbtion in the eye. Therefore, the hydrogels can not be used as a long-term vitreous substitute and can not meet the needs of long-term retina supporting for the treatment of serious diseases such as severe retinal detachment. Hence, how to reduce the degradation of the hydrogel and extend the residence time in vitreous cavity is a very important scientific issue for researchers.
Natural vitreous is surrounded by a "thin film", which is called vitreous cortex. The vitreous cortex structure can be destroyed due to age, trauma and retinopathy. As all the artificial vitreous are directly injected into vitreous cavity, they are easily involved in metabolism and circulation in the eyes. Therefore, it is necessary to find a natural vitreous capsule membrane surrounding the artificial vitreous to restrict the flow in the eye, to avoid or reduce the absorption and deterioration, thereby extending the residence time in vivo.
Therefore, we first propose a new strategy for an alternative natural vitreous, which was the foldable capsular vitreous body (FCVB), mainly constituting ofa balloon, drainage pipes and drainage valve. The balloon is produced by the fine computer simulation vitreous. It connects with the drainage tube, drainage valve, and is made of silicone rubber membrane.The balloon is implanted into the vitreous cavity through a minimally invasive incision, and then the flowing medium, such as saline, silicone oil, hydrogel can be injected into balloon to support the retina.
The foldable capsular vitreous body (FCVB) can avoid the serious defects of the current artificial vitreous. It can well simulate the natural vitreous structure, restore the structural support of retina, as well as the physiological functions of refraction and cell barrier of natural vitreous. And from animal experiments to clinical trials, a series of studies have confirmed that the FCVB tamponade with silicone oil shows excellent biocompatibility, which can effectively360degree support and promote reattachment of the retina, significantly reduce the silicone oil-induced complications such as silicone oil emulsion, secondary glaucoma, corneal degeneration, and so on. However, the FCVB tamponade with silicone oil still can not restore a natureal refractive properties and viscoelastic properties of natural vitreous.
Purpose
This aim of the project was to evaluate a foldable capsular vitreous body (FCVB) injected with polyvinylalcohol (PVA) hydrogel as an artificial vitreous substitute. Different concentrations of PVA hydrogel were crosslinked by irradiation in vitro, then optical, physical, mechanical properties and cytotoxicity were tested to screen a best PVA hydrogel which may be the most proximateto natural vitreous performance. Comprehensive assessment of FCVB injected with PVA hydrogel as a vitreous substitute was received in the rabbit eye model, including optical properties, retinal supporting function and biocompatibility. The novel vitreous substitute of FCVB tamponade with PVA hydrogel can effectively prevent the PVA hydrogel from directly contacting with the retina, and can avoid the PVA hydrogel participating in the intraocular metabolism and flowing into the anterior chamber, as well as reducing the degradation and absorption, extending the residence time of the hydrogel in vitreous cavity, restoring the refractive performance and viscoelastic properties of the natural vitreous.
Materials and Methods
Part one:The preparation of Polyvinyl alcohol (PVA) hydrogel
1. The crosslinking of PVA hydrogel:PVA was purchased from Sigma Aldrich. Different concentrations (1%,3%,7%,w/v) PVA solution were crosslinked by y-irradiation (7kGy, Co60), and1%,3%,7%PVA hydrogels were harvested. Hydrogel was immersed in the double distilled water soaking48h, in order to remove the unreacted crosslinked PVA monomers.
2. Parameters of physical and optical properties:Measuring water content, light transmittance, refractive rate, pH value, and swelling properties of PVA hydrogel.
3. Rheological properties:The rheological analyses were carried out at37±0.1℃on a strain-controlled ARES-RFS rheometer (TA Instruments Inc., New Castle, DE) using a cone and plate geometry of50mm diameter and cone angle of0.04rad. The cross-linked hydrogels were injected onto the plate through a19-gauge needle.
Dynamic strain sweep tests, at a1.0Hz oscillation frequency were performed on the hydrogels to ensure which strain amplitude was in the linear viscoelastic region. The mechanical properties of PVA hydrogels were studied by analyzing the storage modulus (G') and the loss modulus (G") according to frequency of oscillatory shear stress. Dynamic frequency sweep test was conducted at a strain amplitude γ0=1.0%under oscillating frequency ranging from10to0.01Hz.
The relative contribution of the elastic and viscous properties can be quantified by the loss tangent (tan8) which is the ratio of the loss to the storage modulus (tan8=G"/G'). The higher the tan δ, the more liquid-like the sample, with a value of1considered to be a threshold between liquid and gel behavior. Because of the sufficient resilience of the material, when subjected to shear stress, it is also desirable for vitreous substitute. This parameter was estimated by recording the loss tangent. The resilience (R) is an inverse measure of the damping property and usually estimated as R=1-2π tan8.
In a creep experiment, a constant shear stress (σ0) of0.1Pa was imposed to the hydrogel samples for a nominal creep time of500s. In viscoelastic materials the strain (γ) response is linear. The ratio γ/σ0is called creep compliance J(t).
The same analyses were also performed on a usual commercial silicone oil (Oxane5700; Bauch&Lomb, Rochester, NY) and a hyaluronic acid (HA)(Medical Hyaluronan Gel; Iviz, Shangdong, China)
4. Cytotoxicity:The experiment groups included1%,3%, and7%(w/v) PVA hydrogels and a combination of FCVBs that were injected with1%,3%, and7%(w/v) PVA hydrogels. These groups were defined as1%PVA,3%PVA,7%PVA, FCVB+1%PVA, FCVB+3%PVA, and FCVB+7%PVA. The extraction medium was prepared by incubating the varying samples (1%PVA,3%PVA,7%PVA, FCVB+1%PVA, FCVB+3%PVA, and FCVB+7%PVA) with standard culture medium (Dulbecco's modified Eagle's medium with10%fetal calf serum) for72hours at37℃. The extraction medium (200μl) was tested on a monolayer of murine fibroblast L929cells. The murine fibroblast L929cells were seeded in96-well culture plates at a cell density of1.0×103cells/well and fed with standard culture medium for24hours at37℃in a5%CO2atmosphere. Then, cell cultures were incubated with the extraction medium and a negative control (standard culture medium) at the same conditions. After24,48, and72hours of incubation, the cytotoxicity was assessed via a3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbro-mide [MTT, Sigma Aldrich, St. Louis, MO] assay. The absorbance was measured at570nm, and the cytotoxicity was calculated via the following formulae:
cytotoxicity (%)=Optical Density (OD) of sample/Optical Density (OD) of control×100%.
Part two:The research of FCVB injected with PVA hydrogel in vivo
1. Animal Preparation:The rabbit FCVB was made of tailor-made modified liquid silicone rubber. The basic material was Dow Corning Class VI elastomers and the shape was manipulated according to the vitreous parameters of the rabbit by using a computer. The FCVB consists of a vitreous-shaped capsule, tube, and valve, as described in a previous study. The FCVBs were sterilized by heating in double-distilled water at100℃for30min prior to surgery.
All procedures were conducted in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Resolution on the Use of Animals in Vision and Ophthalmic Research. Eighteen New Zealand albino rabbits weighing2.5to3.5kg were divided into three groups:the FCVBs were implanted into the vitreous cavity and then injected with PVA hydrogel and an FCVB (FCVB+PVA group, n=6), PVA hydrogel only (PVA group, n=6), or balanced salt solution (BSS)(BSS group, n=6).
All surgeries were performed with sterile technique in the right eyes of the rabbits. After pupillary dilatation, the rabbits were anesthetized via an intramuscular injection of a mixture of ketamine hydrochloride (30mg/kg) and chlorpromazine hydrochloride (15mg/kg). Under the standard ophthalmic operating microscope, a standard three-port PPV,20-gauge Alcon Accurus vitrectomy system was used. The sclerotomy was created approximately3.5mm posterior to the limbs. A core vitrectomy was performed and followed by a gentle vitrectomy of the peripheral vitreous base. After removing the vitreous as much as possible, PVA hydrogel (1.1ml) or BSS (1.1ml) was directly injected into the vitreous cavity through a19-gauge needle. In the FCVB+PVA group, the lens was cut during vitrectomy for a better long-term observation of the fundus. At the end of the procedure, the FCVB was folded into three petals and implanted into the vitreous cavity through a3mm incision at12o'clock without fluid-air exchange. Then,1.4ml of PVA hydrogel was injected into the capsule through the tube-valve device fixed under the conjunctiva. After surgery, the eyes were treated with tobramycin as antibiotic eyedrops and dexamethasone sodium phosphate as steroid eyedrops. This was done three times a day for2weeks.
2. Postoperative Examinations:A slit lamp and a fundus camera were used to examine and record the anterior segment, ocular media, and fundus at day3,7,14,30,60,90, and180postoperatively. The measurement of intraocular pressure (IOP) with a Tono-Pen was performed at day3,7,14,30,60,90, and180postoperatively. Also, to evaluate the anatomical position of the retina, a B-scan ultrasound was performed at day14,30,60,90, and180postoperatively.
3. Electroretinogram (ERG) tests:A standardized full-field ERG was recorded preoperatively and90days and180days postoperatively on the right eye by using the Roland Ganzfeld system and PC-based signal acquisition and analysis software. The eyes were dilated with0.5%tropicamide and dark-adapted for at least30min. Rabbits were anesthetized as described for surgery. The positive unipolar contact lens was placed on the cornea with methylcellulose (2%). The negative needle electrode was inserted into the subcutaneous in the forehead, and the ground electrode was clipped to the earlobe with some electric gel. The bright flash response was elicited by using the ISCEV standard flash of2.4cds/m2. The photopic ERG was recorded after10min of continuous light adaptation with a background illumination of10cds/m2. The a-wave and b-wave amplitudes were evaluated at different time points.
4. Pathologic Examination:The rabbits were euthanatized via an intramuscular injection of an overdose of ketamine and chlorpromazine (1:1) at90days postoperative (n=3, BSS group; n=3, PVA group; n=3, FCVB+PVA group) and180days postoperative (n=3, BSS group; n=3, PVA group; n=3, FCVB+PVA group). Then, the experimental (right) eyes were harvested from these animals. After enucleation, the PVA hydrogels were removed from the vitreous cavities in order to analyze their elastic and viscous properties. Then, the eyeballs were immediately fixed with10%paraformaldehyde, and they were embedded in paraffin and stained with hematoxylin and eosin (HE) for a light microscope study.
5. Statistical Analysis:All data were analyzed by the SPSS statistical software version13.0(SPSS, Cary, NC, USA). Data were expressed as means±SD. Statistical differences between groups were tested using a one-way analysis of variance (ANOVA). If significance was identified, post hoc analysis with Dunnett was used to confirm the significant changes. A P value of less than0.05was considered statistically significant.
Results
Part one:The preparation of Polyvinyl alcohol (PVA) hydrogel
1. Physical Properties of the Hydrogels:The physical properties of the PVA hydrogels are as follow:the1%PVA hydrogel, with98.9%water content, pH value of7.22, density1.0039, refractive index1.3355, and light transmittance94.8%. The3%PVA hydrogel, with98.1%of water content, pH value7.25, density1.0144, refractive index1.3361, and light transmittance93.2%. The7%PVA hydrogel, with water content93.5%, pH value of7.41, density1.1147, refractive index1.3425, and light transmittance88.2%. After injection, minor fragmentation occurred in only the7%PVA hydrogel, not in the1%and3%PVA hydrogels. All the hydrogels have high water content and are transparent. The1%and3%PVA hydrogels have similar parameters for these physical features and were close to those of the natural human vitreous.
2. Rheological measurements:The oscillatory shear studies were performed on the PVA hydrogels, HA, and silicone oil. The storage modulus (G') represents the elastic or solid-like component, while the loss modulus (G") represents the viscous or liquid-like component. For all the hydrogels, the storage modulus (G') was greater than the loss modulus (G") at all frequencies, which showed "gel-like" behavior. Also, the plots of G' and G" were almost parallel, and there was no crossover in the range of frequencies used. The behaviour is like that of type IV gel. This class of gels would be most suitable for vitreous substitution.
However, for silicon oil, the G" is greater than the G' for all the frequencies analysed, which means that the oil has the appearance of a viscous solution. Meanwhile, the HA behaviour is like type III gel. The G' is generally higher than G", but a G'-G" crossover occurs at low frequencies, which suggest that the HA behaves as an entanglement network and that the sample is mainly viscous rather than elastic.
The values of the human vitreous are similar to those of the porcine vitreous. The average steady state moduli for the porcine vitreous are G'=2.8±0.9Pa and G"=0.7±0.4Pa. Therefore, the7%PVA hydrogel is not suitable as a substitute, due to the fact that its rheological parameters are much higher than porcine rheological parameters.
The loss tangent plots of all samples are recorded as a function of frequency. The lower the loss tangent, the higher the resilience is. Therefore, according to resilience, the hydrogels can be ranked as follows:7%PVA>3%PVA>1%PVA. A material with a high resilience may perform better mechanically in a vitreous cavity.
All the hydrogels were subjected to creep analysis. The compliance of the three analysed hydrogels increases initially and tends to level off and approach a compliance plateau after intermediate periods of time. Therefore, the creep behaviours of these hydrogels are similar to those of very lightly cross-linked amorphous polymers. This behaviour would indicate that there are prominent instantaneous elastic responses followed by substantial retarded elastic responses over time under constant shear stress. The values of the instantaneous elastic deformation of3%PVA is higher than those of1%PVA and7%PVA, indicating that the3%PVA is more elastic.
Therefore, among the three hydrogels, the3%PVA hydrogel would be the best candidate as a vitreous substitute according to the rheological analysis of G', G", loss tangent, and creep behaviour.
Additionally, stable mechanical properties are essential for the hydrogel after injection. The storage modulus G' and the loss modulus G" of the3%PVA hydrogel before and after injection through a-19gauge needle are compared at37℃. The G' value decreased from6.3±0.9to6.1±1.3Pa, and the G" value increased from1.2±0.8to1.3±0.9Pa. The3%PVA hydrogel shows approximately the same rheological behavior before and after the injection, indicating that the injection does not affect the cross-linked structure of the hydrogel.
3. Cytotoxicity tests:The in vitro cytotoxicity of the experimental groups (1%PVA,3%PVA,7%PVA, and FCVB+1%PVA, FCVB+3%PVA, FCVB+7%PVA) was tested on the L929mouse fibroblast cell. No changes in cell morphology, detachment, and membrane lysis were observed in the culture with the tested materials and the negative control. Cell cytotoxicity was evaluated via MTT assay. After24,48, and72hours of incubation, the OD values did not show a significant difference among the experimental groups (P>0.05). Also, there was no significant difference between the experimental groups and the control group (P>0.05). The extractions of the experimental groups induced neither cell viability reduction nor the inhibition of cell growth, resulting in no cytotoxic effects.
To sum up, the3%PVA hydrogel had good rheological properties and good biocompatibility in vitro, so it was selected as a vitreous substitute for further analysis of the long-term biocompatibility and residence time in vivo.
Part two:The research of FCVB injected with PVA hydrogel in vivo
1. Slit lamp:Anterior chamber inflammation was visible in all groups postoperatively. However, the inflammation subsided on the third day after the operation in the BSS group and in the PVA group. Although eyes of FCVB+PVA group had relatively severe inflammation (fibrinous exudation) in the anterior chamber, they recovered within seven days with intensive anti-inflammatory treatments. With the exception of cataracts, no serious complications, such as corneal opacity, keratopathy, or posterior synechia, were observed over180days.
The groups showed varying degrees of lens opacity. In the BSS group, one of six eyes developed cataracts during the180days of observation. In the PVA group, one eye and two eyes developed cataracts in90and180days, respectively. In the FCVB+PVA group, lensectomy was performed during PPV surgery. Our prior study revealed that severe cataracts occurred most frequently in FCVB implantated eyes and that this led to a failure to observe the fundus. In order to avoid lesions from a second surgery, the lens and vitreous were removed at the same time during vitrectomy.
2. Funduscopic examination:Funduscopic examination revealed that there was no evidence of vitritis, uveitis, retinitis, endophthalmitis, vitreous hemorrhage, or retina detachment in the rabbit eyes of all groups. The retina and the optic nerve appeared to be normal. In the BSS group and the FCVB+PVA group, the vitreous cavity remained optically clear on postoperative days90and180. However, in the PVA group, though the eyes showed clarity in their vitreous cavities on postoperative day90, the vitreous cavities appeared to be relatively blurry on postoperative day180due to the complication of cataracts.
3. B-scan ultrasonography:The B-scan ultrasonography showed no retinal detachment in any groups during follow-up. In the FCVB+PVA group, slightly reflective signals of a capsule-like membrane were observed in the vitreous cavity. The FCVB injected with PVA hydrogel was apparently in good contact with the inner retina and can support it well.
4. IOP:A downward trend was observed at postoperative day3in the PVA+FCVB group, which may be due to a leakage of the aqueous humor at the incision for the FCVB implantation before the incision healed. There were upward trends in the IOP after3days in the other two groups, but the IOP reached a plateau level at day7,14,30,60, and90. No statistically significant differences were found in the IOP among the three groups preoperatively or at day7,14,30,60, and90postoperatively, but a significant difference was found in180days. The IOP of the PVA group significantly decreased in180days (P<0.05). This may be due to the fact that some of the PVA hydrogel was dissolved in the vitreous cavities.
5. Electroretinography:Full-field ERG measurements were obtained pre-and post-operatively in all eyes. In the BSS group and PVA groups, the ERG recordings appeared to be similar in terms of the a-wave and b-wave amplitudes before and after operation. There were no significant differences between the eyes of the BSS group and the PVA group in terms of a-or b-wave amplitudes at postoperative day90(P>0.05) and day180(P>0.05). By contrast, in the FCVB+PVA group, a decrease in a-wave and b-wave amplitudes was evident postoperatively, and the differences were significant when compared to the BSS group or PVA group at postoperative day90(P<0.05) and day180(P<0.05).
6. Histological findings:The eyes were enucleated at postoperative day90and day180. In the FCVB+PVA group, a gross examination of eye specimens showed that the capsular wall of the FCVB could fit perfectly with the retina in the vitreous cavity and that the FCVBs injected with3%PVA hydrogel remained transparent and well-rounded. In the PVA group, the vitreous cavities were still filled with3%PVA hydrogel at postoperative day90. Also, the transparency of the3%PVA hydrogel was still very clear, and the viscoelasticity did not seem to have obviously changed. However, the vitreous cavities were filled with3%PVA hydrogel and water-like solution at postoperative day180, and some of the3%PVA hydrogel was dissolved and degenerated. Although the3%PVA hydrogel remained transparent and showed no apparent fragmentation, the viscoelasticity appeared to be poorer than it was before implantation.
The histological examination of H&E-stained retinal sections from the BSS group and PVA group showed that the integrity of the retinal layers was good and that no loss of tissue was evident. There was no evidence of pathological changes, such as deformations, degeneration, or inflammation. In the FCVB+PVA group eyes terminated on postoperative day90, the H&E-stained sections displayed a normal retinal morphology. In eyes terminated at day180, retinal disorder was seen. The retinas displayed an aggregation of the inner nuclear layer and the outer nuclear layer and a thinning of the ganglion cells layer. No signs of inflammation were seen. Additionally, in all three groups, there were no pathological changes in other parts of the eye, including the cornea and ciliary body.
7. Oscillatory shear measurements after in vivo experiments:After sacrifice and enucleation, the3%PVA hydrogel of the vitreous cavity in the PVA group and the FCVB+PVA group was collected and subjected to oscillatory shear measurements, as described before. The mechanical spectrum of the hydrogels. In the FCVB+PVA group, on postoperative day90and day180, the G' and G" values remained similar to those before surgery. In the PVA group, though a mild decrease in the G' and G" values was observed at postoperative day90, a conspicuous decrease was observed at postoperative day180as compared to preoperative values. The G' decreased from6.1±1.3to3.9±0.8Pa and G" decreased from1.3±0.9to0.8±0.5Pa. This indicated that the3%PVA hydrogel had undergone obvious biodegradation in the vitreous cavity after a180-day retention.
Conclusions
3%PVA hydrogel has similar optical, physical and rheological properties to natural vitreous, and shows good biocompatibility. After a long-term (180days) tamponade in the vitreous cavity, the FCVB injected with3%PVA hydrogel as a vitreous substitute reveals good optical, mechanical properties and biocompatibility, which can effectively support the retina and maintain the anatomical structure of the retina as well as effectively avoiding degradation of the PVA hydrogeland elongating the residence time. This new approach may develop into a valuable tool to improve the stability performance of PVA hydrogel as a vitreous substitute and to extend the application function of FCVB for long-term implantation in vitreous cavity.
玻璃体是一种透明的不可再生的凝胶体,其主要由99%的水与1%的无机盐、胶原和透明质酸等构成,胶原纤维呈三维网状结构,其上附着透明质酸粘多糖,后者能与水分子结合,从而使玻璃体呈凝胶状。玻璃体的主要生理功能是支撑视网膜、眼屈光、细胞屏障和营养等。由于自然玻璃体的不可再生性,因此,当发生玻璃体视网膜疾病时,如：外伤导致的视网膜脱离、外伤性增生性玻璃体视网膜病变(T-PVR)、增生性玻璃体视网膜病变(PVR)、增生性糖尿病视网膜病变(PDR)、严重眼内炎等等,需行玻璃体切割手术治疗,术中切除自然玻璃体后必须填充合适的人工玻璃体替代物,修复眼损伤,支撑视网膜,重建视功能,防止眼球萎缩。
目前,研究者们对玻璃体替代物的研究甚多,临床常用的主要有空气、惰性气体、重水、硅油、重硅油等。使用的方式均为直接注入至玻璃体腔,与视网膜直接接触,利用其表面张力而达到支撑视网膜的效果。以上除硅油外,均为短期应用。然而,长期的硅油填充,容易导致一系列的术后并发症,如并发性白内障、继发性青光眼、角膜变性、硅油乳化、甚至可移行至视网膜下和视神经,造成视神经纤维的髓鞘脱失,导致视力永久丧失。因此,颇为亟待寻找一种具有良好生物相容性,无严重并发症,并能长期填充的人工玻璃体。
水凝胶因具有良好的生物相容性、光学性能、减震性,能模拟自然玻璃体的特性,而被誉为是人工玻璃体最好的候选者,从上世纪九十年代以来一直是国内外的研究热点。主要有聚乙烯醇[poly(vinyl alcohol), PVA]水凝胶、聚乙二醇[polyethylene glycol), PEG]水凝胶、聚丙烯酸[poly(acrylic acid), PAA]水凝胶和聚丙烯酰胺[polyacrylamide, PAM]水凝胶等。研究者们可体外通过改变水凝胶的合成过程,来满足自然玻璃体的物理性能,然后将其注入体内。也可以将液态的高分子物质注射入玻璃体腔后进行原位交联而形成水凝胶,这就可避免注射过程中对水凝胶结构的破坏。同时,水凝胶还可以负载药物,作为眼内药物释放的载体。作为短期的玻璃体替代物,水凝胶在动物实验眼中应用并没有明显的术后并发症。但由于其注入玻璃体腔后,与视网膜直接接触,使得水凝胶参与眼内代谢和循环,导致在眼内容易被降解和吸收。因此,仍然不能作为长期的玻璃体替代物,不能满足对治疗严重的视网膜脱离等疾病需要长期顶压的要求。所以,如何减少或阻止水凝胶在眼内的降解吸收,延长水凝胶在玻璃体腔的停留时间,使之能成为一种长期填充的人工玻璃体是眼科研究一个非常重要的科学问题。
自然玻璃体有一层薄膜包围,此薄膜属于解剖学玻璃体皮质范畴。由于年龄、外伤和视网膜玻璃体病变等因素,薄膜结构可遭到破坏,受此启发,我们研究总结发现目前人工玻璃体的研究方法是直接注射,都没有解决人工玻璃体和眼内组织直接接触导致的各种问题。需要研究一个能像自然玻璃体一样的囊膜包绕人工玻璃体,限制人工玻璃体的眼内流动,避免或减少被吸收和变质,从而延长人工玻璃体的使用时间。
因此,我们首次提出一个替代自然玻璃体的新策略-折叠式人工玻璃体(FCVB),它主要由球囊、引流管和引流阀组成。球囊部是通过计算机精细模拟人眼玻璃体制作而成,并且通过引流管与引流阀相连接,由能在体内应用20年以上的硅橡胶薄膜制成。通过微创切口将球囊部植入到玻璃体腔,随后注入流动介质,如生理盐水、硅油、水凝胶等,形成玻璃体形状的薄膜球囊支撑视网膜、充填眼球。
折叠式人工玻璃体避免了目前人工玻璃体的严重缺点,可以精细模拟自然玻璃体的结构,恢复自然玻璃体的视网膜支撑、眼屈光、细胞屏障等主要生理功能。并通过从动物实验、标准制定到临床试验的系列研究证实,折叠式人工玻璃体填充硅油在眼内长期填充后显示出良好的生物相容性,有效的360。全方位支撑视网膜,促进脱离视网膜的复位,能显著减少硅油诱导的并发症如硅油乳化、继发性青光眼、角膜变性等。但是,由于折叠式人工玻璃体填充硅油后无法恢复自然玻璃体的屈光性能,没有恢复自然玻璃体所拥有的机械减震和粘弹性能。
目的
因此,结合水凝胶的国际研究进展,本课题提出应用折叠式人工玻璃体填充聚乙烯醇(PVA)水凝胶重建自然玻璃体的设想。利用辐照法交联得到不同浓度的PVA水凝胶,体外检测光学、物理、机械性能及细胞毒性等,筛选最接近于人自然玻璃体性能的PVA水凝胶；在兔眼模型中全面评估FCVB填充PVA水凝胶作为玻璃体替代物的光学物理性能、视网膜支撑功能和生物相容性等。本研究旨在探讨FCVB填充PVA水凝胶作为玻璃体替代物,具有良好的生物相容性,能够避免PVA水凝胶与视网膜的直接接触,阻止PVA水凝胶参与眼内代谢和循环,减少PVA水凝胶的降解和吸收,延长水凝胶在玻璃体腔内的停留时间,恢复自然玻璃体的屈光、视网膜支撑和粘弹性能,从而达到重建自然玻璃体的目的。
材料和方法
第一部分：聚乙烯醇(PVA)水凝胶的制备及其性能与生物相容性研究
1.PVA水凝胶的制备：聚乙烯醇(PVA)购于Sigma Aldrich公司。配制不同浓度(1%、3%、7%,质量-体积分数)PVA溶液,采用γ-射线(Co60)辐照交联得到1%PVA水凝胶、3%PVA水凝胶、7%PVA水凝胶。将水凝胶分别浸入双蒸水中浸泡48h,以除去未交联PVA单体。
2.物理与光学性能：测量充分溶胀后的PVA水凝胶的比重、含水率、透光率、屈光率、pH值、溶胀性能等参数。
3.流变性能：PVA水凝胶经充分溶胀后,应用ARES-RFS流变仪对水凝胶行动态应变扫描测试、粘弹性能和蠕变性能测试。测量温度为37±0.1℃。动态应变扫描测试：在振荡频率1Hz条件下,确定线性粘弹区间的应变振幅后；在该应变振幅下,检测水凝胶在振荡频率范围为0.01-10Hz时储存模量(G')和损耗模量(G")的动态变化。蠕变测试,在恒定剪切应力σ0=0.1Pa条件下,检测水凝胶随着剪切应力时间的延长(500s),其应变(力反应的变化趋势。用同样方法测量硅油和透明质酸酸钠的流变性能。通过对比选取其中一种与自然玻璃体参数最为相似的PVA水凝胶。
4.体外细胞毒性试验：采用噻唑蓝比色法(MTT法)测定PVA水凝胶对L929鼠成纤维细胞生长抑制作用。制备待测样本浸泡液：样本分为6组(1%PVA、3%PVA、7%PVA、FCVB+1%PVA、FCVB+3%PVA、FCVB+7%PVA),分别将样本放入DMEM培养基浸泡24h、48h、72h,收集浸泡液备用。取对数生长期细胞,接种于96孔培养板,每孔加100μL细胞悬液(每孔5000个细胞)细胞贴壁后,加入不同样本的浸泡液,以培养液作为对照。将培养板置入37℃、5%C02培养箱中,分别培养24h、48h、72h。显微镜下观察细胞形态和行MTT比色检测,酶标仪于波长570nm下测定OD值并计算抑制率,评价样品的细胞毒性。
5.统计学分析：采用SPSS13.0统计软件进行分析,所有计量资料用均数±标准差(X±SD)表示。MTT比色试验中,不同时间点及不同组OD值用单个重复测量因素方差分析,P<0.05认为具有统计学显著差异。
第二部分：折叠式人工玻璃体(FCVB)填充PVA水凝胶重建自然玻璃体的体内研究
1.动物手术：选取体重2.5-3kg新西兰白兔18只,所有实验操作严格遵守ARVO关于实验动物使用的规定。分为三组：PVA水凝胶组(PVA组)、FCVB+PVA水凝胶组(FCVB+PVA组)、平衡盐液组(BSS组)。右眼行常规三通道玻璃体切割术,切除玻璃体,术中填充PVA水凝胶6眼、填充FCVB+PVA水凝胶6眼、填充BSS溶液6眼。术后常规予典必殊滴眼液、普南扑灵滴眼液和百力特滴眼液滴术眼抗炎预防感染治疗。
2.术后观察：术后3、7、14、30、60、90、180天,常规行裂隙灯、直接眼底镜、眼压和眼底照相检查,观察术眼前节和眼底情况；术后14、30、60、90和180天行术眼B超检查了解玻璃体腔和眼底视网膜解剖位置情况；术后90、180天,分别行双眼ERG检查,评估玻璃体替代物植入后对视网膜功能的影响。
3.眼球病理检测：于术后90天(PVA组,3只；FCVB+PVA组,3只；BSS组,3只)和180天(PVA组,3只；FCVB+PVA组,3只；BSS组,3只),用过量利多卡因麻醉处死兔子,剜出眼球,马上取出并收集眼内PVA水凝胶后,迅速将眼球迅速放入固定液中,固定眼球,按常规方法做石蜡切片光学显微镜、HE染色检查。
4.PVA水凝胶性能检测：将眼内填充后的PVA水凝胶分别行透光率、屈光率和流变学检查。方法同前。
5.统计学分析：采用SPSS13.0统计软件进行分析,所有计量资料用均数±标准差(X±SD)表示。眼压用单个重复测量因素方差分析,ERG振幅值用单因素方差分析(One-way ANOVA),方差齐时采用S-N-K法进行多重比较,方差不齐时采用近似F检验Welch法进行校正,采用Dunnett T3法进行多重比较。P<0.05认为具有统计学显著差异。
结果
第一部分：聚乙烯醇(PVA)水凝胶的制备及其性能与生物相容性研究
1.光学及物理性能：1%PVA水凝胶的含水率为98.9%,屈光指数为1.3355,透光率为94.8%,pH值为7.22,比重为1.0039；3%PVA水凝胶的含水率为98.1%,屈光指数为1.3361,透光率为93.2%,pH值为7.25,比重为1.0144；7%PVA水凝胶的含水率为93.5%,屈光指数为1.3425,透光率为88%,pH值为7.41,比重为1.1147；自然玻璃体的含水率为98-99%,屈光指数为1.3345-1.3348,透光率为>90%,pH值为7.0-7.4,比重为1.0053-1.0089。
2.流变性能：三种浓度PVA水凝胶储存模量(G’)均大于损耗模量(G"),表明三种物质均为有粘弹性能的凝胶体,且其弹性性能大于粘性性能。而硅油表现为损耗模量(G")大于储存模量(G'),提示其粘性性能大于弹性性能,因此主要表现为粘性液体的状态。1%PVA:G'为3.2±1.1Pa,G"为0.8±0.5Pa；3%PVA:G'为6.1±1.3Pa,G"为1.3±0.9Pa:7%PVA:G'为106.5±18.6Pa,G''为18.3±12.8Pa；硅油：G'为2.4±1.6Pa,G"为44.8±28.3Pa;透明质酸：G'为155.2±147.2Pa,G"为92.9±49.1Pa。自然玻璃体：G'为2.8±0.9Pa,G"为0.7±0.4Pa
在顺应性方面,良好的顺应性使得在玻璃体腔内可以拥有更为良好的机械性能,7%PVA>3%PVA>1%PVA;在蠕变方面,它代表的是迅时弹性形变的能力和柔韧性,3%PVA>7%PVA>1%PVA。综合以上光学、物理和流变性能,可以得出3%PVA水凝胶的性能更为接近自然玻璃体,因此选择3%PVA水凝胶作为FCVB填充物进行体内的进一步研究。
3.细胞毒性：6组(1%PVA、3%PVA、7%PA、FCVB+1%PVA、 FCVB+3%PVA、FCVB+7%PVA)材料样品浸泡提取液与L929细胞共培养24、48及72h后,光镜下观察细胞形态和结构均无显著变化。MTT比色法测定OD值,提示在24、48、72h三个时间点,1%PVA、3%PVA、7%PVA组及FCVB+1%PvA、FCVB+3%PVA、FCVB+7%PVA组与对照组,OD值比较均无显著统计学差异(P>0.05)。所以,结果提示FCVB填充PVA水凝胶后或单纯PVA水凝胶均无明显细胞毒性。
第二部分：折叠式人工玻璃体(FCVB)填充PVA水凝胶重建自然玻璃体的体内研究
1.裂隙灯：术后3天,PVA组及BSS组均出现了不同程度结膜水肿、充血和轻度前房炎症反应,至术后7天症状全部消失；而FCVB+PVA组在术后7天前房仍出现少量炎性细胞,但未见瞳孔后粘连及纤维素样渗出,给予积极抗炎防感染治疗后,到术后14天结膜水肿、充血及前房炎症逐渐消退。在随后长达半年的随访观察,三组白兔术眼的眼前节均为出现明显异常,但是PVA组和BSS组有术眼出现不同程度的晶体混浊,术后90天时白内障发生率：PVA组为16.7%(1/6)；术后180天时白内障发生率：PVA组为33.3%(1/3),BSS组为16.7%(1/6)；由于先前研究发现兔眼植入FCVB后白内障的发生率较高,考虑本研究观察时间较长,白内障严重后会影响玻璃体腔和眼底视网膜等组织的观察,为避免二次手术,本研究中FCVB+PVA组采取行玻璃体切割手术的同时行晶体切除术。
2.眼底检查：术后14天,FCVB+PVA组随着炎症不断消退,眼底可隐约见视盘和血管,至术后30天,眼底能清晰可见,直至观察终点术后180天,玻璃体腔仍然保持透明,能清晰看到眼底视网膜、血管、视盘等结构。在PVA组,术后14天时能清晰看到眼底,然而随着白内障的发展和进一步加重,术后90天和180天,眼底逐渐变得模糊,但是仍可见眼底视盘、血管、视网膜等大体结构。BSS组在180天的观察期内,眼底均可清晰可见。随访观察期内三组术眼均未见有玻璃体出血、混浊、玻璃体视网膜增殖和视网膜脱离等情况发生。
3.眼压：术后第3天,FCVB+PVA组眼压呈现下降趋势,与术前比较有显著统计学差异(P<0.05),但到术后7天,眼压逐渐上升至正常范围,与术前比较无显著统计学差异(P>0.05),至术后14天、30天、60天、90天、180天,眼压一直维持在正常范围,与术前比较无显著统计学差异(P>0.05)。在PVA组和BSS组,术后3天眼压出现明显的升高,与术前相比有显著统计学差异(P<0.05),但到术后7天,眼压逐渐降低至正常范围,与术前比较无显著统计学差异(P>0.05),至术后14天、30天、60天、90天,眼压维持较平稳,与术前比较无显著统计学差异(P>0.05)。但是在术后180天,PVA组眼压与术前相比呈显著下降趋势(P<0.05)。
4.眼部B超：在术后整个180天的随访观察期内,三组兔眼眼部B超均未显示有玻璃体混浊、玻璃体腔出血、增殖或视网膜脱离、脉络膜脱离等声像。FCVB+PVA组眼底B超提示FCVB囊壁与视网膜相贴,良好的支撑视网膜。
5.眼部电生理：全视网膜电图(ERG)检查提示：FCVB+PVA组,术后90天和术后180天,a波和b波峰值均比术前下降,差异具有统计学意义(P<0.05)。在PVA组和BSS组,术后90和180天a波和b波峰值与术前变化比较差异无统计学意义(P>0.05)。
6.眼球病理：FCVB+PVA组,术后90、180天剜出眼球,大体解剖观察可见眼球充实饱满,FCVB填充PVA水凝胶后能良好的支撑视网膜,取出FCVB球囊内的PVA水凝胶仍然透明和维持较好的粘弹性。PVA组,术后90天摘出眼球饱满充实,取出眼内PVA水凝胶亦保持透明和一定的粘弹性；但是在术后180天时,摘出的眼球外观轻度萎缩,取出眼内PVA水凝胶虽然透明,但是明显被降解和吸收,间中混有较多的水样物质,粘弹性明显减弱。取出眼球固定后,石蜡固定切片,HE染色,提示：除FCVB+PVA组在术后180天视网膜出现结构紊乱、神经纤维层变薄,神经节细胞结构破坏等情况外,其余均未见明显结构异常包括角膜、睫状体、视网膜等。
7.光学性能与流变学检查：术后90天,FCVB+PVA组与PVA组填充后的PVA水凝胶其光学和流变性能和填充前相比无显著改变。在FCVB+PVA组,PVA水凝胶填充180天后,其光学与流变性能和填充前相比仍然无明显改变,PVA水凝胶无明显降解和吸收。然而在PVA组,PVA水凝胶填充180天后,其折光率和流变性能较前发生明显改变,折光率降低为1.3121,储存模量(G')从6.1±1.3降至3.9±0.8Pa,损耗模量(G")从1.3±0.9降至0.8±0.5Pa,粘弹性能明显降低,提示经180天眼内填充后PVA水凝胶显著被降解和吸收。
结论
1.3%PVA水凝胶具有与自然玻璃体相似的光学、物理和流变性能,拥有良好的生物相容性,能很好的模拟自然玻璃体的性能。
2. FCVB填充3%PVA水凝胶作为一个新的玻璃体替代物,经兔眼玻璃体腔内的长期(180天)填充后,发现其具有良好的光学性能、机械性能和生物相容性,能够安全有效、全方位的支撑视网膜,维持视网膜的解剖结构；FCVB能有效的发挥生物屏障的功能,阻止PVA水凝胶的快速降解和吸收,延长PVA水凝胶在玻璃体内的填充时间,为寻找一种理想的玻璃体替代物提供新的思路和方法。
Background
The vitreous body is a clear, non-renewable gel body, which is mainly constituted by the99%water and1%inorganic salts, collagen and hyaluronic acid. Collagen fibers are three-dimensional mesh structure, attached by hyaluronic mucopolysaccharide, which can be combined with water molecules, so that the vitreous is substantially gelled. The physiological functions of vitreous body are supporting the retina, refraction, cell barriers and nutrition in eyes. Due to the non-renewable properties of natural vitreous body, when vitreoretinal diseases occur, such as:the trauma caused retinal detachment, traumatic proliferative vitreoretinopathy (T-PVR), proliferative vitreoretinopathy (PVR), proliferative diabetic retinopathy (PDR) and so on, vitrectomy surgery is needed. After intraoperative resection of natural vitreous body, suitable artificial vitreous substitutes must be filled into vitreous cavities to repair the damage, supporting retina, reconstructing visual function to prevent atrophy of eyeball.
Currently, the vitreous substitutes commonly used in clinical include air, inert gas, heavy water, silicone oil, heavy silicone oil and so on. All of them are directly injected into the vitreous cavity, directly contacting with the retina, to support retina by the surface tension. The others are short-term tamponade in vivo in addition to silicone oil. However, after a long-term tamponade in vitreous cavity, a series of post-operative complications are likely to occur, such as cataract, secondary glaucoma, corneal degeneration, silicone oil emulsification and even migrate to subretina or optic nerve, causing demyelination of the optic nerve fibers, resulting in permanent loss of vision. Therefore, it is quite urgent to find an ideal vitreous substitute with good biocompatibility without serious complications for long-term tamponade.
Hydrogel is known as one of the best artificial vitreous candidates because of its good biocompatibility, optical property and shock absorption performances. It can mimic the characteristics of natural vitreous, and has been a hot research focus at home and abroad since the1990s. Hydrogel mainly includes PVA [poly (vinyl alcohol)] hydrogel, PEG [poly (ethylene glycol)] hydrogel, PAA [poly (acrylic acid)] hydrogel, PAM [poly acrylamide] hydrogel, and so on. To meet the physical properties of natural vitreous, researchers have been trying to change different synthesis process of the hydrogel in vitro, and then injected into the vitreous cavity. Liquid polymeric substance may also be injected into the vitreous cavity which is crosslinked in situ to form hydrogel in vitreous cavity. In this way, it can avoid the structure destruction during injection of the hydrogel. Additionally, the hydrogel can load drugs, acting as a release vector of drug within the eye. There were no significant postoperative complications after a short-term tamponade in animal experiments. Because all the substitutes are directly injected into the vitreous cavity, directly contacting with the retina, the hydrogels are also easily involved in the metabolism and circulation within the eye, resulting in rapidly degradation and absorbtion in the eye. Therefore, the hydrogels can not be used as a long-term vitreous substitute and can not meet the needs of long-term retina supporting for the treatment of serious diseases such as severe retinal detachment. Hence, how to reduce the degradation of the hydrogel and extend the residence time in vitreous cavity is a very important scientific issue for researchers.
Natural vitreous is surrounded by a "thin film", which is called vitreous cortex. The vitreous cortex structure can be destroyed due to age, trauma and retinopathy. As all the artificial vitreous are directly injected into vitreous cavity, they are easily involved in metabolism and circulation in the eyes. Therefore, it is necessary to find a natural vitreous capsule membrane surrounding the artificial vitreous to restrict the flow in the eye, to avoid or reduce the absorption and deterioration, thereby extending the residence time in vivo.
Therefore, we first propose a new strategy for an alternative natural vitreous, which was the foldable capsular vitreous body (FCVB), mainly constituting ofa balloon, drainage pipes and drainage valve. The balloon is produced by the fine computer simulation vitreous. It connects with the drainage tube, drainage valve, and is made of silicone rubber membrane.The balloon is implanted into the vitreous cavity through a minimally invasive incision, and then the flowing medium, such as saline, silicone oil, hydrogel can be injected into balloon to support the retina.
The foldable capsular vitreous body (FCVB) can avoid the serious defects of the current artificial vitreous. It can well simulate the natural vitreous structure, restore the structural support of retina, as well as the physiological functions of refraction and cell barrier of natural vitreous. And from animal experiments to clinical trials, a series of studies have confirmed that the FCVB tamponade with silicone oil shows excellent biocompatibility, which can effectively360degree support and promote reattachment of the retina, significantly reduce the silicone oil-induced complications such as silicone oil emulsion, secondary glaucoma, corneal degeneration, and so on. However, the FCVB tamponade with silicone oil still can not restore a natureal refractive properties and viscoelastic properties of natural vitreous.
Purpose
This aim of the project was to evaluate a foldable capsular vitreous body (FCVB) injected with polyvinylalcohol (PVA) hydrogel as an artificial vitreous substitute. Different concentrations of PVA hydrogel were crosslinked by irradiation in vitro, then optical, physical, mechanical properties and cytotoxicity were tested to screen a best PVA hydrogel which may be the most proximateto natural vitreous performance. Comprehensive assessment of FCVB injected with PVA hydrogel as a vitreous substitute was received in the rabbit eye model, including optical properties, retinal supporting function and biocompatibility. The novel vitreous substitute of FCVB tamponade with PVA hydrogel can effectively prevent the PVA hydrogel from directly contacting with the retina, and can avoid the PVA hydrogel participating in the intraocular metabolism and flowing into the anterior chamber, as well as reducing the degradation and absorption, extending the residence time of the hydrogel in vitreous cavity, restoring the refractive performance and viscoelastic properties of the natural vitreous.
Materials and Methods
Part one:The preparation of Polyvinyl alcohol (PVA) hydrogel
1. The crosslinking of PVA hydrogel:PVA was purchased from Sigma Aldrich. Different concentrations (1%,3%,7%,w/v) PVA solution were crosslinked by y-irradiation (7kGy, Co60), and1%,3%,7%PVA hydrogels were harvested. Hydrogel was immersed in the double distilled water soaking48h, in order to remove the unreacted crosslinked PVA monomers.
2. Parameters of physical and optical properties:Measuring water content, light transmittance, refractive rate, pH value, and swelling properties of PVA hydrogel.
3. Rheological properties:The rheological analyses were carried out at37±0.1℃on a strain-controlled ARES-RFS rheometer (TA Instruments Inc., New Castle, DE) using a cone and plate geometry of50mm diameter and cone angle of0.04rad. The cross-linked hydrogels were injected onto the plate through a19-gauge needle.
Dynamic strain sweep tests, at a1.0Hz oscillation frequency were performed on the hydrogels to ensure which strain amplitude was in the linear viscoelastic region. The mechanical properties of PVA hydrogels were studied by analyzing the storage modulus (G') and the loss modulus (G") according to frequency of oscillatory shear stress. Dynamic frequency sweep test was conducted at a strain amplitude γ0=1.0%under oscillating frequency ranging from10to0.01Hz.
The relative contribution of the elastic and viscous properties can be quantified by the loss tangent (tan8) which is the ratio of the loss to the storage modulus (tan8=G"/G'). The higher the tan δ, the more liquid-like the sample, with a value of1considered to be a threshold between liquid and gel behavior. Because of the sufficient resilience of the material, when subjected to shear stress, it is also desirable for vitreous substitute. This parameter was estimated by recording the loss tangent. The resilience (R) is an inverse measure of the damping property and usually estimated as R=1-2π tan8.
In a creep experiment, a constant shear stress (σ0) of0.1Pa was imposed to the hydrogel samples for a nominal creep time of500s. In viscoelastic materials the strain (γ) response is linear. The ratio γ/σ0is called creep compliance J(t).
The same analyses were also performed on a usual commercial silicone oil (Oxane5700; Bauch&Lomb, Rochester, NY) and a hyaluronic acid (HA)(Medical Hyaluronan Gel; Iviz, Shangdong, China)
4. Cytotoxicity:The experiment groups included1%,3%, and7%(w/v) PVA hydrogels and a combination of FCVBs that were injected with1%,3%, and7%(w/v) PVA hydrogels. These groups were defined as1%PVA,3%PVA,7%PVA, FCVB+1%PVA, FCVB+3%PVA, and FCVB+7%PVA. The extraction medium was prepared by incubating the varying samples (1%PVA,3%PVA,7%PVA, FCVB+1%PVA, FCVB+3%PVA, and FCVB+7%PVA) with standard culture medium (Dulbecco's modified Eagle's medium with10%fetal calf serum) for72hours at37℃. The extraction medium (200μl) was tested on a monolayer of murine fibroblast L929cells. The murine fibroblast L929cells were seeded in96-well culture plates at a cell density of1.0×103cells/well and fed with standard culture medium for24hours at37℃in a5%CO2atmosphere. Then, cell cultures were incubated with the extraction medium and a negative control (standard culture medium) at the same conditions. After24,48, and72hours of incubation, the cytotoxicity was assessed via a3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbro-mide [MTT, Sigma Aldrich, St. Louis, MO] assay. The absorbance was measured at570nm, and the cytotoxicity was calculated via the following formulae:
cytotoxicity (%)=Optical Density (OD) of sample/Optical Density (OD) of control×100%.
Part two:The research of FCVB injected with PVA hydrogel in vivo
1. Animal Preparation:The rabbit FCVB was made of tailor-made modified liquid silicone rubber. The basic material was Dow Corning Class VI elastomers and the shape was manipulated according to the vitreous parameters of the rabbit by using a computer. The FCVB consists of a vitreous-shaped capsule, tube, and valve, as described in a previous study. The FCVBs were sterilized by heating in double-distilled water at100℃for30min prior to surgery.
All procedures were conducted in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Resolution on the Use of Animals in Vision and Ophthalmic Research. Eighteen New Zealand albino rabbits weighing2.5to3.5kg were divided into three groups:the FCVBs were implanted into the vitreous cavity and then injected with PVA hydrogel and an FCVB (FCVB+PVA group, n=6), PVA hydrogel only (PVA group, n=6), or balanced salt solution (BSS)(BSS group, n=6).
All surgeries were performed with sterile technique in the right eyes of the rabbits. After pupillary dilatation, the rabbits were anesthetized via an intramuscular injection of a mixture of ketamine hydrochloride (30mg/kg) and chlorpromazine hydrochloride (15mg/kg). Under the standard ophthalmic operating microscope, a standard three-port PPV,20-gauge Alcon Accurus vitrectomy system was used. The sclerotomy was created approximately3.5mm posterior to the limbs. A core vitrectomy was performed and followed by a gentle vitrectomy of the peripheral vitreous base. After removing the vitreous as much as possible, PVA hydrogel (1.1ml) or BSS (1.1ml) was directly injected into the vitreous cavity through a19-gauge needle. In the FCVB+PVA group, the lens was cut during vitrectomy for a better long-term observation of the fundus. At the end of the procedure, the FCVB was folded into three petals and implanted into the vitreous cavity through a3mm incision at12o'clock without fluid-air exchange. Then,1.4ml of PVA hydrogel was injected into the capsule through the tube-valve device fixed under the conjunctiva. After surgery, the eyes were treated with tobramycin as antibiotic eyedrops and dexamethasone sodium phosphate as steroid eyedrops. This was done three times a day for2weeks.
2. Postoperative Examinations:A slit lamp and a fundus camera were used to examine and record the anterior segment, ocular media, and fundus at day3,7,14,30,60,90, and180postoperatively. The measurement of intraocular pressure (IOP) with a Tono-Pen was performed at day3,7,14,30,60,90, and180postoperatively. Also, to evaluate the anatomical position of the retina, a B-scan ultrasound was performed at day14,30,60,90, and180postoperatively.
3. Electroretinogram (ERG) tests:A standardized full-field ERG was recorded preoperatively and90days and180days postoperatively on the right eye by using the Roland Ganzfeld system and PC-based signal acquisition and analysis software. The eyes were dilated with0.5%tropicamide and dark-adapted for at least30min. Rabbits were anesthetized as described for surgery. The positive unipolar contact lens was placed on the cornea with methylcellulose (2%). The negative needle electrode was inserted into the subcutaneous in the forehead, and the ground electrode was clipped to the earlobe with some electric gel. The bright flash response was elicited by using the ISCEV standard flash of2.4cds/m2. The photopic ERG was recorded after10min of continuous light adaptation with a background illumination of10cds/m2. The a-wave and b-wave amplitudes were evaluated at different time points.
4. Pathologic Examination:The rabbits were euthanatized via an intramuscular injection of an overdose of ketamine and chlorpromazine (1:1) at90days postoperative (n=3, BSS group; n=3, PVA group; n=3, FCVB+PVA group) and180days postoperative (n=3, BSS group; n=3, PVA group; n=3, FCVB+PVA group). Then, the experimental (right) eyes were harvested from these animals. After enucleation, the PVA hydrogels were removed from the vitreous cavities in order to analyze their elastic and viscous properties. Then, the eyeballs were immediately fixed with10%paraformaldehyde, and they were embedded in paraffin and stained with hematoxylin and eosin (HE) for a light microscope study.
5. Statistical Analysis:All data were analyzed by the SPSS statistical software version13.0(SPSS, Cary, NC, USA). Data were expressed as means±SD. Statistical differences between groups were tested using a one-way analysis of variance (ANOVA). If significance was identified, post hoc analysis with Dunnett was used to confirm the significant changes. A P value of less than0.05was considered statistically significant.
Results
Part one:The preparation of Polyvinyl alcohol (PVA) hydrogel
1. Physical Properties of the Hydrogels:The physical properties of the PVA hydrogels are as follow:the1%PVA hydrogel, with98.9%water content, pH value of7.22, density1.0039, refractive index1.3355, and light transmittance94.8%. The3%PVA hydrogel, with98.1%of water content, pH value7.25, density1.0144, refractive index1.3361, and light transmittance93.2%. The7%PVA hydrogel, with water content93.5%, pH value of7.41, density1.1147, refractive index1.3425, and light transmittance88.2%. After injection, minor fragmentation occurred in only the7%PVA hydrogel, not in the1%and3%PVA hydrogels. All the hydrogels have high water content and are transparent. The1%and3%PVA hydrogels have similar parameters for these physical features and were close to those of the natural human vitreous.
2. Rheological measurements:The oscillatory shear studies were performed on the PVA hydrogels, HA, and silicone oil. The storage modulus (G') represents the elastic or solid-like component, while the loss modulus (G") represents the viscous or liquid-like component. For all the hydrogels, the storage modulus (G') was greater than the loss modulus (G") at all frequencies, which showed "gel-like" behavior. Also, the plots of G' and G" were almost parallel, and there was no crossover in the range of frequencies used. The behaviour is like that of type IV gel. This class of gels would be most suitable for vitreous substitution.
However, for silicon oil, the G" is greater than the G' for all the frequencies analysed, which means that the oil has the appearance of a viscous solution. Meanwhile, the HA behaviour is like type III gel. The G' is generally higher than G", but a G'-G" crossover occurs at low frequencies, which suggest that the HA behaves as an entanglement network and that the sample is mainly viscous rather than elastic.
The values of the human vitreous are similar to those of the porcine vitreous. The average steady state moduli for the porcine vitreous are G'=2.8±0.9Pa and G"=0.7±0.4Pa. Therefore, the7%PVA hydrogel is not suitable as a substitute, due to the fact that its rheological parameters are much higher than porcine rheological parameters.
The loss tangent plots of all samples are recorded as a function of frequency. The lower the loss tangent, the higher the resilience is. Therefore, according to resilience, the hydrogels can be ranked as follows:7%PVA>3%PVA>1%PVA. A material with a high resilience may perform better mechanically in a vitreous cavity.
All the hydrogels were subjected to creep analysis. The compliance of the three analysed hydrogels increases initially and tends to level off and approach a compliance plateau after intermediate periods of time. Therefore, the creep behaviours of these hydrogels are similar to those of very lightly cross-linked amorphous polymers. This behaviour would indicate that there are prominent instantaneous elastic responses followed by substantial retarded elastic responses over time under constant shear stress. The values of the instantaneous elastic deformation of3%PVA is higher than those of1%PVA and7%PVA, indicating that the3%PVA is more elastic.
Therefore, among the three hydrogels, the3%PVA hydrogel would be the best candidate as a vitreous substitute according to the rheological analysis of G', G", loss tangent, and creep behaviour.
Additionally, stable mechanical properties are essential for the hydrogel after injection. The storage modulus G' and the loss modulus G" of the3%PVA hydrogel before and after injection through a-19gauge needle are compared at37℃. The G' value decreased from6.3±0.9to6.1±1.3Pa, and the G" value increased from1.2±0.8to1.3±0.9Pa. The3%PVA hydrogel shows approximately the same rheological behavior before and after the injection, indicating that the injection does not affect the cross-linked structure of the hydrogel.
3. Cytotoxicity tests:The in vitro cytotoxicity of the experimental groups (1%PVA,3%PVA,7%PVA, and FCVB+1%PVA, FCVB+3%PVA, FCVB+7%PVA) was tested on the L929mouse fibroblast cell. No changes in cell morphology, detachment, and membrane lysis were observed in the culture with the tested materials and the negative control. Cell cytotoxicity was evaluated via MTT assay. After24,48, and72hours of incubation, the OD values did not show a significant difference among the experimental groups (P>0.05). Also, there was no significant difference between the experimental groups and the control group (P>0.05). The extractions of the experimental groups induced neither cell viability reduction nor the inhibition of cell growth, resulting in no cytotoxic effects.
To sum up, the3%PVA hydrogel had good rheological properties and good biocompatibility in vitro, so it was selected as a vitreous substitute for further analysis of the long-term biocompatibility and residence time in vivo.
Part two:The research of FCVB injected with PVA hydrogel in vivo
1. Slit lamp:Anterior chamber inflammation was visible in all groups postoperatively. However, the inflammation subsided on the third day after the operation in the BSS group and in the PVA group. Although eyes of FCVB+PVA group had relatively severe inflammation (fibrinous exudation) in the anterior chamber, they recovered within seven days with intensive anti-inflammatory treatments. With the exception of cataracts, no serious complications, such as corneal opacity, keratopathy, or posterior synechia, were observed over180days.
The groups showed varying degrees of lens opacity. In the BSS group, one of six eyes developed cataracts during the180days of observation. In the PVA group, one eye and two eyes developed cataracts in90and180days, respectively. In the FCVB+PVA group, lensectomy was performed during PPV surgery. Our prior study revealed that severe cataracts occurred most frequently in FCVB implantated eyes and that this led to a failure to observe the fundus. In order to avoid lesions from a second surgery, the lens and vitreous were removed at the same time during vitrectomy.
2. Funduscopic examination:Funduscopic examination revealed that there was no evidence of vitritis, uveitis, retinitis, endophthalmitis, vitreous hemorrhage, or retina detachment in the rabbit eyes of all groups. The retina and the optic nerve appeared to be normal. In the BSS group and the FCVB+PVA group, the vitreous cavity remained optically clear on postoperative days90and180. However, in the PVA group, though the eyes showed clarity in their vitreous cavities on postoperative day90, the vitreous cavities appeared to be relatively blurry on postoperative day180due to the complication of cataracts.
3. B-scan ultrasonography:The B-scan ultrasonography showed no retinal detachment in any groups during follow-up. In the FCVB+PVA group, slightly reflective signals of a capsule-like membrane were observed in the vitreous cavity. The FCVB injected with PVA hydrogel was apparently in good contact with the inner retina and can support it well.
4. IOP:A downward trend was observed at postoperative day3in the PVA+FCVB group, which may be due to a leakage of the aqueous humor at the incision for the FCVB implantation before the incision healed. There were upward trends in the IOP after3days in the other two groups, but the IOP reached a plateau level at day7,14,30,60, and90. No statistically significant differences were found in the IOP among the three groups preoperatively or at day7,14,30,60, and90postoperatively, but a significant difference was found in180days. The IOP of the PVA group significantly decreased in180days (P<0.05). This may be due to the fact that some of the PVA hydrogel was dissolved in the vitreous cavities.
5. Electroretinography:Full-field ERG measurements were obtained pre-and post-operatively in all eyes. In the BSS group and PVA groups, the ERG recordings appeared to be similar in terms of the a-wave and b-wave amplitudes before and after operation. There were no significant differences between the eyes of the BSS group and the PVA group in terms of a-or b-wave amplitudes at postoperative day90(P>0.05) and day180(P>0.05). By contrast, in the FCVB+PVA group, a decrease in a-wave and b-wave amplitudes was evident postoperatively, and the differences were significant when compared to the BSS group or PVA group at postoperative day90(P<0.05) and day180(P<0.05).
6. Histological findings:The eyes were enucleated at postoperative day90and day180. In the FCVB+PVA group, a gross examination of eye specimens showed that the capsular wall of the FCVB could fit perfectly with the retina in the vitreous cavity and that the FCVBs injected with3%PVA hydrogel remained transparent and well-rounded. In the PVA group, the vitreous cavities were still filled with3%PVA hydrogel at postoperative day90. Also, the transparency of the3%PVA hydrogel was still very clear, and the viscoelasticity did not seem to have obviously changed. However, the vitreous cavities were filled with3%PVA hydrogel and water-like solution at postoperative day180, and some of the3%PVA hydrogel was dissolved and degenerated. Although the3%PVA hydrogel remained transparent and showed no apparent fragmentation, the viscoelasticity appeared to be poorer than it was before implantation.
The histological examination of H&E-stained retinal sections from the BSS group and PVA group showed that the integrity of the retinal layers was good and that no loss of tissue was evident. There was no evidence of pathological changes, such as deformations, degeneration, or inflammation. In the FCVB+PVA group eyes terminated on postoperative day90, the H&E-stained sections displayed a normal retinal morphology. In eyes terminated at day180, retinal disorder was seen. The retinas displayed an aggregation of the inner nuclear layer and the outer nuclear layer and a thinning of the ganglion cells layer. No signs of inflammation were seen. Additionally, in all three groups, there were no pathological changes in other parts of the eye, including the cornea and ciliary body.
7. Oscillatory shear measurements after in vivo experiments:After sacrifice and enucleation, the3%PVA hydrogel of the vitreous cavity in the PVA group and the FCVB+PVA group was collected and subjected to oscillatory shear measurements, as described before. The mechanical spectrum of the hydrogels. In the FCVB+PVA group, on postoperative day90and day180, the G' and G" values remained similar to those before surgery. In the PVA group, though a mild decrease in the G' and G" values was observed at postoperative day90, a conspicuous decrease was observed at postoperative day180as compared to preoperative values. The G' decreased from6.1±1.3to3.9±0.8Pa and G" decreased from1.3±0.9to0.8±0.5Pa. This indicated that the3%PVA hydrogel had undergone obvious biodegradation in the vitreous cavity after a180-day retention.
Conclusions
3%PVA hydrogel has similar optical, physical and rheological properties to natural vitreous, and shows good biocompatibility. After a long-term (180days) tamponade in the vitreous cavity, the FCVB injected with3%PVA hydrogel as a vitreous substitute reveals good optical, mechanical properties and biocompatibility, which can effectively support the retina and maintain the anatomical structure of the retina as well as effectively avoiding degradation of the PVA hydrogeland elongating the residence time. This new approach may develop into a valuable tool to improve the stability performance of PVA hydrogel as a vitreous substitute and to extend the application function of FCVB for long-term implantation in vitreous cavity.
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