Abstract
目的
制备地塞米松/米诺环素(dexamethasone/minocycline，Dex/Mino)脂质体(liposome，lipo)修饰的聚醚醚酮(polyetheretherketone，PEEK)，探究功能化PEEK在体内应用时是否能有效预防感染且促进新骨再生，实现生理骨整合。
方法
利用聚多巴胺(polydopamine，pDA)涂层作中间介质，将Dex/Mino脂质体修饰到PEEK表面，通过荧光脂质体接枝进行定性和定量检测，评价脂质体是否成功共价修饰在该表面。分别建立小鼠皮下植入感染模型和比格犬股骨植入模型，通过Micro-CT及苏木精-伊红(hematoxylin-eosin，HE)染色分析，评价Dex/Mino脂质体修饰的PEEK的体内生物活性。
结果
荧光脂质体接枝定性和定量结果显示，pDA功能化修饰组的红色荧光强度强于非功能化修饰组(P < 0.05)，由于pDA涂层的存在，脂质体成功接枝于PEEK表面并在其表面均匀分布。小鼠皮下PEEK片植入24 h后，HE染色结果显示，与纯PEEK组相比，PEEK-Dex/Mino lipo组的炎性细胞数量较低(P < 0.05)，即感染程度较低，提示脂质体中Mino的释放在体内能有效地预防感染。比格犬股骨植入8周后，Micro-CT和HE染色结果显示，在PEEK-Dex/Mino lipo组中，新骨形成比纯PEEK组多，且牢固地结合在功能化修饰的PEEK表面，沿着PEEK界面延伸，提示脂质体中Dex的释放在体内有效地刺激和引导了新骨再生。
结论
Dex/Mino脂质体修饰提高了惰性PEEK的生物活性，功能化PEEK具有增强的骨整合能力(预防感染和促进新骨再生)，其作为牙科/骨科替代修复材料极具临床应用前景。
Keywords: 脂质体, 聚醚醚酮, 骨整合, 抑菌, 地塞米松, 米诺环素
Abstract
Objective
To develop dexamethasone plus minocycline-loaded liposomes (Dex/Mino liposomes) and apply them to improve bioinert polyetheretherketone (PEEK) surface, which could prevent post-operative bacterial contamination, enhance ossification for physiologic osseointegration, and finally reduce implant failure rates.
Methods
Dex/Mino liposomes were covalently grafted onto the PEEK surface using polydopamine (pDA) coating as a medium. Confocal laser scanning microscopy was used to confirm the binding of fluorescently labeled liposomes onto the PEEK substrate, and a microplate reader was used to semiquantitatively measure the average fluorescence intensity of fluorescently labeled liposome-decorated PEEK surfaces. Moreover, the mouse subcutaneous infection model and the beagle femur implantation model were respectively conducted to verify the bioactivity of Dex/Mino liposome-modified PEEK in vivo, by means of micro computed tomography (micro-CT) and hematoxylin and eosin (HE) staining analysis.
Results
The qualitative and quantitative results of fluorescently labeled liposomes showed that, the red fluorescence intensity of the PEEK-pDA-lipo group was stronger than that of the PEEK-NF-lipo group (P < 0.05); the liposomes were successfully and uniformly decorated on the PEEK surfaces due to the pDA coating. After mouse subcutaneous implantation of PEEKs for 24 hours, HE staining results showed that the number of inflammatory cells in the PEEK-Dex/Mino lipo group were lower than that in the inert PEEK group (P < 0.05), indicating a lower degree of infection in the test group. These results suggested that the Mino released from the liposome-functionalized surface provided an effective bacteriostasis in vivo. After beagle femoral implantation of PEEK for 8 weeks, micro-CT results showed that the PEEK-Dex/Mino lipo group newly formed more continuous bone when compared with the inert PEEK group; HE staining results showed that more new bones were formed in the PEEK-Dex/Mino lipo group than in the inert PEEK group, which were firmly bonded to the functionalized PEEK surface and extended along the PEEK interface. These results suggested that the Dex released from the liposome-functionalized surface induced effective bone regeneration in vivo.
Conclusion
Dex/Mino liposome modification enhanced the bioactivity of inert PEEK, the functionalized PEEK with enhanced antibacterial and osseointegrative capacity has great potential as an orthopedic/dental implant material for clinical application.
Keywords: Liposomes, Polyetheretherketone, Osseointegration, Bacteriostasis, Dexamethasone, Minocycline
目前应用较为广泛的骨替代修复材料主要包括纯钛及钛合金等医用金属材料和聚醚醚酮(polyetheretherketone，PEEK)等高分子材料，其中PEEK具有多种优越的性能，尤其是其弹性模量与人体皮质骨的弹性模量较接近，可以降低应力屏蔽现象，避免可能的骨损伤[1-2]。但PEEK是生物惰性材料，在体内不能发挥生物活性，进而影响了植入物-骨界面的骨整合。影响植入物-骨界面骨整合的因素很多，当前研究认为主要包括植入物表面的促成骨活性和感染[3-4]。因此，为了拓宽PEEK在骨科、口腔科等领域的临床应用前景，必须提高惰性PEEK的成骨活性，同时使其能预防细菌污染。既往文献报道，最常用的方法是通过物理或化学方法制备生物活性涂层使PEEK功能化，许多涂层(包括羟基磷灰石、生物活性小分子、磷酸钙、钛等)在增强PEEK植入物的骨整合方面具有实用价值[5-7]，但这些方法仍然存在许多亟待解决的问题，包括化学步骤复杂耗时、涂层易降解、涂层与基底粘接不良等。
聚多巴胺(polydopamine，pDA)涂层是一种易于操作的表面改性方法。通过在弱碱性水环境中简单浸泡方式，多巴胺几乎可以在所有材料表面(无需预处理)形成结构稳定的pDA薄膜，而且pDA的邻苯二酚结构可以进一步连接含氨基或巯基的生物活性分子，进而实现材料表面的二次修饰，该方法已被广泛用于各种生物材料的功能化[8-9]。但pDA不适合直接固定进入细胞内发挥作用的生物活性小分子，如米诺环素(minocycline，Mino; 一种广谱四环素类抗生素)和地塞米松(dexamethasone，Dex; 一种具有促成骨作用的糖皮质激素)[10-11]。目前，克服这一限制的策略主要是使用纳米递送系统，如脂质体(liposome，lipo)，其可携带、运输和缓慢释放生物活性小分子到细胞质中[12]。
结合以上研究背景，本研究提出PEEK表面功能化改性方案(图 1)：用脂质体作为药物运输载体同时包载Dex和Mino，然后进一步利用多巴胺的自聚合及迈克尔加成反应(Michael addition reaction)，将Dex/Mino脂质体共价接枝在PEEK表面; 先对功能化的PEEK进行荧光表征，再将其植入体内，分别建立小鼠皮下植入感染模型和比格犬股骨植入模型，评价该表面修饰方法是否有效地提高了PEEK表面的抑菌和促成骨活性，为进一步将该材料用于临床提供依据。
图 1.
Dex/Mino脂质体功能化修饰的PEEK表面的制备及其体内骨整合性能(包括抑菌和促成骨活性)评价的示意图
Schematic illustration of the preparation of Dex/Mino liposome-decorated PEEK through pDA coating, as well as its bacteriostasis and osseointegration in vivo for load-bearing bone repairing
PEEK, polyetheretherketone; pDA, polydopamine; Dex, dexamethasone; Mino, minocycline; S. mutans, Streptococcus mutans.
1. 材料与方法
1.1. 制备Dex/Mino脂质体
Dex/Mino脂质体按照薄膜分散法制备[13-14]。按照摩尔比例1.85 : 1.00 : 0.15 : 0.25精密称取相应的二棕榈酰磷脂酰胆碱、胆固醇、二硬脂酰基磷脂酰乙醇胺-聚乙二醇-氨基和Dex于茄形瓶中，按照1 : 1(体积比)加入甲醇和氯仿的混合溶剂并摇动使溶质溶解; 使用75 r/min的旋转蒸发仪在47 ℃恒温水浴中减压干燥彻底除去有机溶剂，瓶内壁形成一层均匀的脂膜。取适量的水化液(120 mmol/L无水乙酸钠和无水氯化钙的混合液，pH=7.9)加入茄形瓶中，于47 ℃恒温水浴中超声水化，直至内壁脂膜完全脱落形成悬液; 迅速将所得脂质体悬液依次通过孔径为220 nm和100 nm的滤器挤出数次，即得到具有浓度梯度的Dex脂质体，使用0.9%(质量分数)氯化钠缓冲液过夜透析。透析后的Dex脂质体与适量Mino储液(药脂比为1 : 10)混合，50 ℃条件下振摇茄形瓶30 min使Mino载入脂质体水相中，即得到Dex/Mino脂质体，使用磷酸盐缓冲液过夜透析后4 ℃保存备用。
1.2. 制备Dex/Mino脂质体修饰的PEEK
将纯PEEK片(直径10 mm，长度1 mm)和纯PEEK种植体(直径4 mm，长度7 mm)依次放置于丙酮、无水乙醇和去离子水中超声清洗各2 h，50 ℃干燥后待用。将多巴胺粉末加入pH=8.5的Tris缓冲液(10 mmol/L)中混匀得到多巴胺溶液(2 g/L)，然后将纯PEEK片/纯PEEK种植体放入多巴胺溶液中，在37 ℃条件下反应18 h，摇床设置为70 r/min，超声清洗3次后将样品命名为PEEK-pDA。37 ℃条件下将PEEK-pDA在Dex/Mino脂质体悬液(1 g/L)中静置浸泡24 h，轻轻清洗3次后将样品命名为PEEK-Dex/Mino lipo。
1.3. 荧光脂质体修饰的PEEK表面的定性及定量检测
本实验在避光条件下完成。分别将纯PEEK和PEEK-pDA片浸泡于1.0 g/L的荧光脂质体溶液中24 h，轻轻清洗3次，得到的样品命名为PEEK非功能化(non-functionalized，NF)脂质体(PEEK-NF-lipo)和PEEK-pDA-lipo。使用激光共聚焦显微镜拍摄样品表面脂质体分布情况; 加入甲醇400 μL溶解化学接枝的脂质体(70 r/min，5 min)，取100 μL到96孔板中，使用酶标仪检测PEEK表面脂质体的荧光接枝密度。
1.4. 小鼠皮下植入感染模型建立
1.4.1. 实验动物
雄性C57BL/6小鼠(6~8周龄)，由北京维通利华实验动物技术有限公司提供，在无特定病原体(specific pathogen free，SPF)级环境下饲养。本研究得到了北京大学动物伦理委员会的批准(批准号：LA2015149)。
1.4.2. 变形链球菌菌液准备
变形链球菌(UA159)由北京大学口腔医院中心实验室微生物平台提供。培养方法：于-80 ℃冰箱取出甘油冻存菌，溶化后与Todd-Hewitt(TH)液体培养基混匀，使用螺旋接种仪将菌液复苏于脑心浸液(brain heart infusion，BHI)固体培养基上，将培养皿倒置培养48 h，挑取一个单克隆于TH液体培养基(1 mL)中继续培养24 h备用。
1.4.3. 手术植入及石蜡切片染色
实验分两组，每组两只小鼠。将小鼠称重，使用1%(质量分数)戊巴比妥钠腹腔注射使其麻醉; 将小鼠俯卧位固定，脱毛使皮肤充分暴露，常规消毒; 使用组织剪剪开小鼠皮肤，钝性分离形成皮下囊袋; 使用加样枪在两组PEEK片表面分别接种10 μL变形链球菌(1×108 CFU/mL); 将表面接种了细菌的PEEK片置于皮下，使用角针缝合皮肤; 术后将小鼠放于37 ℃保温台上，观察小鼠麻醉后苏醒状态。术后24 h，腹腔注射1%(质量分数)戊巴比妥钠处死小鼠后取样，组织块于4%(质量分数)多聚甲醛中固定后流水冲洗，脱水透明，浸蜡并包埋形成蜡块; 使用石蜡切片机切片后进行苏木精-伊红(hematoxylin and eosin，HE)染色。
1.5. 比格犬股骨植入模型建立
1.5.1. 实验动物
三只健康的雄性比格犬，1.5年龄，体质量(11.2±0.6) kg，由北京玛斯生物技术有限公司提供，在普通级环境下饲养。本研究得到了北京大学动物伦理委员会的批准(批准号：LA2018226)。
1.5.2. 比格犬股骨内种植体的植入
比格犬称重，使用3%(质量分数)戊巴比妥钠胫前静脉注射使其全身麻醉。将比格犬侧卧位固定于恒温手术台上，脱毛使皮肤充分暴露，常规消毒铺巾; 切开皮肤，切口处局部注射含肾上腺素的利多卡因，小心分离肌肉、血管，充分暴露股骨; 使用高速涡轮机和直径为4 mm的钻针备洞，洞深达到7 mm，备洞过程中使用生理盐水降温; 将样本置于准备好的洞中，每个股骨随机植入2~3个种植体，两组分别植入8个种植体; 依次用圆针和角针严密对位缝合肌肉和皮肤。术后及时观察比格犬麻醉苏醒状态，连续3天肌肉注射10 U/kg青霉素钠，留意观察比格犬的伤口愈合情况，若出现红肿或感染等症状则及时处理，第7天拆线。
1.5.3. Micro-CT分析及硬组织切片染色
手术8周后，胫前静脉注射3%(质量分数)戊巴比妥钠处死，获取股骨组织标本，将其放置于10%(质量分数)中性福尔马林溶液中充分固定; 利用Micro-CT扫描股骨样本，使用Micro-CT分析软件重建三维的骨质结构。Micro-CT完成后，修剪样本，流水清洗，使用梯度乙醇对样本脱水，将股骨样本先后浸泡于渗透液Ⅰ、Ⅱ和Ⅲ中，然后置入包埋液中固定，使用硬组织修片机切割修整样本，切片后进行HE染色。
1.6. 统计学分析
使用Origin 8.0统计软件对实验结果进行分析处理; 数据均以均数±标准差表示，组间比较采用t检验，P < 0.05为差异有统计学意义。
2. 结果
2.1. PEEK表面荧光脂质体接枝定性及定量分析
本实验使用荧光检测来评价脂质体是否共价修饰于PEEK表面，图 2为代表性的荧光图像及其相对应的脂质体接枝强度定量。荧光标记的脂质体同时附着于PEEK-NF-lipo和PEEK-pDA-lipo上，PEEK-pDA-lipo组的红色荧光强度强于PEEK-NF-lipo组，并在其表面均匀分散。定量结果进一步证实，由于pDA涂层的存在，更多荧光标记的脂质体被共价固定在PEEK表面，说明脂质体成功修饰于PEEK表面并在其表面均匀分布。
图 2.
PEEK-NF-lipo组和PEEK-pDA-lipo组表面的荧光定性及定量分析(n=11)
Qualitative and quantitative fluorescence analysis of PEEK-NF-lipo group and PEEK-pDA-lipo group (n=11)
NF, non-functionalized; lipo, liposome. Other abbreviations as in Figure 1. *P < 0.05.
2.2. 小鼠皮下PEEK片植入后抑菌作用的评价
本实验将Dex/Mino脂质体修饰的PEEK(表面接种变形链球菌菌液)植入C57BL/6小鼠皮下，评估其体内抗菌活性和组织学反应。通过HE染色评估小鼠体内组织学反应发现，两组样品周围的皮下组织中都有炎性细胞(图 3)，但与纯PEEK组相比，PEEK-Dex/Mino lipo组的炎性细胞数量较低，说明纯PEEK组的感染严重程度高于功能化PEEK组，提示Dex/Mino脂质体修饰的PEEK在体内可有效地预防感染。
图 3.
小鼠皮下植入24 h后的组织切片HE染色及炎性细胞定量(n=5)
HE staining and inflammatory cell quantification of mouse after subcutaneous implantation for 24 h (n=5)
A, HE staining of mouse after subcutaneous implantation for 24 h; B, quantification of inflammatory cells in Figure A (×20). Abbreviations as in Figure 1 and 2. *P < 0.05.
2.3. 比格犬股骨内PEEK植入后骨整合能力的评价
本研究建立了一个比格犬股骨植入模型，将功能化PEEK植入股骨骨髓腔(图 4A、B)，评价改性PEEK表面的骨整合能力。术后8周采用Micro-CT技术来评估骨样本，可见纯PEEK周围的新生骨是不连续的、间断的(图 4C)，而Dex/Mino脂质体修饰的PEEK周围则形成更多连续的、致密的新骨(图 4D); 纯PEEK组的新生骨容积比例(骨体积/总体积，bone volume/total volume，BV/TV)为0.09，PEEK-Dex/Mino lipo组的BV/TV值为0.27。上述结果表明，与纯PEEK相比，Dex/Mino脂质体修饰的PEEK显著促进了新骨形成。HE染色结果显示，纯PEEK界面有新骨碎片，且与新骨组织的间隙较大; PEEK-Dex/Mino lipo组新骨形成比纯PEEK组多，且牢固地结合在功能化修饰的PEEK表面，沿着PEEK界面延伸(图 5)，与Micro-CT结果一致。以上结果提示，Dex/Mino脂质体的修饰加速了骨沉积，促进了骨融合，Dex/Mino脂质体修饰的PEEK能更好地与宿主骨结合，促进新骨形成。
图 4.
功能化PEEK植入比格犬股骨8周后的Micro-CT分析
Micro-CT analysis of functionalized PEEK after 8 weeks of Beagle's femur implantation
A, a PEEK implant; B, macroscopic image of a beagle's femur containing implants; C, micro-CT 3D reconstruction images of PEEK; D, micro-CT 3D reconstruction images of PEEK-Dex/Mino lipo.
图 5.
功能化PEEK植入比格犬股骨8周后的横向示意图及HE染色分析
Schematic illustration of the transverse perspective and HE staining analysis of functionalized PEEK after 8 weeks of Beagle's femur implantation
Abbreviations as in Figure 1 and 2.
3. 讨论
由感染、创伤、炎症、骨肿瘤等原因导致的骨缺损通常需要使用骨替代修复材料进行手术植入修复[15-18]。决定骨替代修复材料植入成功与否的关键是植入物-骨界面的骨整合，材料的表面性质决定了植入物与体内周围组织整合的最终能力[19-20]。为了实现植入后的最佳骨整合，在不破坏PEEK众多优点的前提下，采用表面改性方法增强PEEK表面的生物活性是首选途径[1]。本研究以pDA涂层作中间介质，将Dex/Mino脂质体修饰到PEEK表面，使其表面功能化，荧光脂质体接枝定性及定量检测提示脂质体已成功共价修饰在该表面，而且在该表面均匀分布。
由于植入物表面与周围组织的界面易受细菌侵袭，如果感染得不到控制，可能会导致术后植入失败，因此植入物表面的抑菌作用至关重要[21]。细菌在植入物界面上的初始粘附是感染的一个关键因素，也是生物膜形成的一个关键步骤，考虑到植入物的持久使用，在术后植入初期阻断细菌对植入生物材料的粘附至关重要[22-23]。本研究通过建立小鼠皮下植入感染模型，评估功能化PEEK在体内的抑菌活性及组织学反应。HE染色结果显示，与纯PEEK组相比，PEEK-Dex/Mino lipo组的炎性细胞浸润程度较轻，即感染程度较轻，说明Dex/Mino脂质体修饰提高了惰性PEEK的抗感染活性，Mino的释放在体内更有效地预防了感染发生。
骨整合会指引成骨细胞在植入物表面定植，合成细胞外骨基质，最终形成新骨[24-25]。研究体内组织细胞对Dex/Mino脂质体修饰的PEEK的反应是骨整合的重要指标之一，同时与本研究的PEEK材料是否能够成为生物医学植入物密切相关。本研究通过建立比格犬股骨植入模型，评估功能化PEEK的体内新骨生成及骨整合能力。Micro-CT及组织学分析结果显示，与纯PEEK组相比，PEEK-Dex/Mino lipo组促进更多新骨生成，而且功能化PEEK与新生骨牢固结合。既往研究指出，Dex修饰的纳米颗粒或纳米纤维可促进体外成骨相关蛋白表达及体内钙化骨形成[26-27]，提示Dex/Mino脂质体涂层修饰PEEK后，脂质体中Dex的释放必然促进骨髓腔中成骨细胞的生长及新骨再生。经Dex/Mino脂质体修饰后，Dex的释放在体内更有效地刺激和引导了新骨再生。生物惰性PEEK周围的新骨形成增加，进一步促进了PEEK与骨之间的骨化，提示Dex/Mino脂质体修饰的PEEK更有利于提高体内骨整合。
综上所述，Dex/Mino脂质体修饰的PEEK在体内具有有效的抑菌和促进新骨再生的能力，作为牙科和骨科替代修复材料具有很大的临床应用潜力。
Funding Statement
国家自然科学基金(81571814)
Supported by the National Natural Science Foundation of China (81571814)
Contributor Information
许 晓 (Xiao XU), Email: 15266257389@163.com.
魏 世成 (Shi-cheng WEI), Email: sc-wei@pku.edu.cn.
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