CN115137875B - 一种高效的双相磷酸钙涂层方法 - Google Patents
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Abstract
本发明提供一种高效的双相磷酸钙涂层方法,通过采用高浓度(4.5×)的过饱和磷酸钙溶液(SCPS)进行涂层,在钛片上形成了更厚的连续涂层,且涂层中含有二水磷酸二钙和羟基磷灰石,该涂层对蛋白质FITC‑BSA的包封率明显提高,且在酸性条件下释放的Ca2+更少,同时能以更少的SCPS溶液体积,更短的涂层时间,实现具有高成品率和蛋白质包封率的更高效的涂层,从而在临床应用中大幅降低成本,非常有希望应用于生物医学领域的成骨植入材料。
Description
技术领域
本发明属于生物涂层材料制备领域,具体而言,涉及一种具有高成品率和蛋白质包封率的高效双相磷酸钙涂层方法。
背景技术
钛及钛合金以其力学性能、生物相容性以及耐腐蚀方面的优良特性已用于人体硬组织植入物如人工关节、骨折固定器等领域。但将其直接植入到人体中后,由于钛及其表面自然生成的氧化膜是生物惰性的,在植入早期很难与组织形成连接并促进新生骨在表面的生成,从而表现出与骨结合力低、生物活性差、愈合时间长等问题。为解决这些问题,在其表面进行生物活性涂层处理是一种行之有效的方法。
钙-磷酸盐(CaP)涂层又称双相磷酸钙涂层,由于其类骨成分、良好的生物相容性和可调节各种成骨过程的能力,是最广泛使用的表面修饰方法,以促进骨内植入体和骨缺损填充材料的骨整合能力和骨传导性。目前,制备CaP涂层主要有两种方法:1)物理沉积技术和2)湿化学技术。物理沉积技术包括各种热过程,如真空等离子喷涂和悬浮等离子喷涂。物理沉积技术的主要局限性是非生理的涂层条件(>1000℃)使其不可能加入能够促进成骨的生物活性物质。因此,发展新型的湿化学沉积技术来制备生物活性CaP涂层是生物材料研究领域的一个新趋势。
仿生涂层技术最初是在20世纪90年代提出的。通过将生物材料浸入离子浓度、pH值和温度接近人类血浆的模拟体液(SBF)中,可形成致密、均匀、典型的低结晶度磷灰石。这种生理条件使大量生物活性物质能够与仿生涂层共沉淀,并保持其生物活性。然而,最初的仿生涂层工艺的实施由于其耗时(通常为14天)和依赖于生物材料的官能团成核而受到限制。Taset等人通过将SBF浓度提高10倍(10×SBF),成功地将涂层过程显著加快到数小时。然而,SBF沉积依赖于生物材料功能基团的问题仍然没有解决。
2000年,Klaas教授及其同事研发了一种双相仿生涂层方法:1)先通过在5×SBF溶液中孵育形成无定形CaP基底层,随后在过饱和磷酸钙(supersaturated calcium-phosphate solution,SCPS)中孵育形成CaP结晶层。这种双相仿生方法能够在3天内形成CaP晶体涂层。此外,这种涂层可应用于具有不同三维几何形状、表面结构和表面化学性质的多种生物材料,如金属(钛合金)、无机材料(TCP和Bio Oss)和高分子材料(天然衍生的胶原蛋白和合成聚乳酸-羟基乙酸)。这种仿生涂层的广泛适用性在很大程度上归功于无定形CaP基底层,它可以形成一种机械嵌合机制,而不是与活性化学基团进行化学反应。无定形CaP层是后续CaP结晶层生长所必需的。该CaP结晶层可用于共沉淀、负载和缓慢释放各种生物活性物质,特别是骨诱导生长因子骨形态发生蛋白2(BMP-2)。与表面吸附的BMP-2相比,加入涂层的BMP-2的成骨诱导效率显著提高,这可能与涂层的控释特性有关。
然而,由于过饱和磷酸钙(SCPS)离子浓度较低,昂贵的生物活性物质的包封率较低(约21%),阻碍了其在生物医学领域的应用。Yu等试图通过调整基质表面积与改良SBF(m-SBF)的体积比,仅使用1ml m-SBF即将包封率提高到90%。然而,他们并没有报道最终的涂层厚度,而一定的涂层厚度对于储备足够量的生物活性药物和确保足够的给药时间以支持骨形成是很重要的。
因此急需找到一种成品率和蛋白质包封率更高,涂层效率更高、厚度更厚、效果更好的双相磷酸钙(CaP)涂层方法,从而满足是生物医学领域对骨内植入生物材料的生物活性涂层需要。
发明内容
为解决现有技术中存在的问题,本发明提供一种具有高成品率和蛋白质包封率的高效双相磷酸钙涂层方法,通过采用高浓度(4.5×)的过饱和磷酸钙溶液(SCPS)进行涂层,在钛片上形成了更厚的连续涂层,且涂层中含有二水磷酸二钙和羟基磷灰石,该涂层对荧光标记的模型蛋白质FITC-BSA的包封率明显提高,且在酸性条件下释放的Ca2+更少。本发明提供的改良的涂层工艺能以更少的SCPS溶液体积,更短的涂层时间,实现涂层更厚,生物活性物质包封率更高,非常有希望应用于生物医学领域的成骨植入材料。
一方面,本发明提供了一种双相磷酸钙涂层,所述涂层含有二水磷酸二钙和羟基磷灰石。
本发明提供的双相磷酸钙涂层是二水磷酸二钙(DCPD)和羟基磷灰石的复合物。DCPD具有生物相容性、生物可降解性和骨传导性,可转化为无水磷酸二钙(DCPA)(pH<6)、磷酸八钙(OCP)(pH=6-7)或沉淀羟基磷灰石(pHAp)(pH>7)。在医学上,DCPD可用于CaP骨水泥的制备。研究发现,DCPD在体内既可以转化为羟基磷灰石,也可以降解并被骨取代。
本发明提供的含有二水磷酸二钙(DCPD)和羟基磷灰石的涂层,具有更坚实的结构,并且沉积了更多的仿生CAP,能携带更多的活性蛋白,具有抗破骨吸收的耐酸性能,在酸性条件下释放的Ca2+更少,在体内降解更加缓慢,性能明显优于仅含有羟基磷灰石的涂层。
另一方面,本发明提供了一种的制备方法,包括以下步骤:
(1)将钛片浸入5×SBF中,制备CaP基底层;
(2)取出,干燥后,浸入1倍浓度以上的SCPS中。
本发明提供的双相磷酸钙涂层的制备,主要通过采用更高浓度的过饱和磷酸钙溶液(SCPS)进行涂层,从而实现涂层效果的改良。
进一步地,步骤(2)所述的1倍浓度以上的SCPS为4.5×SCPS。
本发明所述的5×SBF是指5倍浓度的SBF;4.5×SCPS是指4.5倍浓度的SCPS;1×SCPS是指1倍浓度的SCPS。
发明人经过大量的研究实验,令人惊喜地发现,通过采用更高浓度的过饱和磷酸钙溶液(SCPS)进行涂层,尤其是采用4.5×SCPS时,可以实现更高成品率和蛋白质包封率的高效双相磷酸钙涂层,且涂层为二水磷酸二钙(DCPD)和羟基磷灰石的复合物,更坚实,在酸性条件下释放的Ca2+更少,在体内降解更加缓慢,明显优于1×SCPS的涂层效果。
进一步地,其中,5×SBF、1×SCPS和4.5×SCPS的无机离子组成如下表所示:
进一步地,步骤(2)为:取出,干燥后,浸入4.5×SCPS中,室温下孵育5h以上。
进一步地,步骤(1)为:将钛片浸入5×SBF中,37℃孵育24h,制备CaP基底层。
CaP基底层是一种无定型CaP,可用于共沉淀、负载和缓慢释放各种生物活性物质,是后续CaP结晶层生长所必需的,可为后面的晶体形成提供结晶核。
进一步地,所述钛片的长和宽都是4mm,厚度为1mm。
进一步地,步骤(2)所述的4.5×SCPS的体积为0.1-6ml。
研究证明,4.5×SCPS的体积与涂层的厚度呈双相关系,当4.5×SCPS的体积在0.1–1ml范围内时,涂层厚度快速增加,当4.5×SCPS的体积达到1ml以上时,尤其是在在较高的溶液体积(2–6ml)时,涂层厚度增加相对缓慢。
在4.5×SCPS体积为0.1ml时,既能获得晶体结构清晰的涂层,通过场发射扫描电镜已观察不到无定型CaP;0.3ml的4.5×SCPS就能形成连续的涂层,1ml的4.5×SCPS已经能够形成非常厚的涂层。可见通过4.5×SCPS可以采用更少的体积实现更高性能的涂层。
再一方面,本发明提供了4.5×SCPS在用于制备双相磷酸钙涂层方面的用途,所述双相磷酸钙涂层含有二水磷酸二钙和羟基磷灰石。
再一方面,本发明提供了4.5×SCPS在提高双相磷酸钙涂层的成品率、蛋白质包封率、涂层厚度中的一项或多项的用途。
本发明具有以下有益效果:
(1)通过4.5×SCPS制得了成品率更高、蛋白质包封率更高、涂层厚度更厚的双相磷酸钙涂层,涂层质量和产量都得到显著提升;
(2)通过4.5×SCPS可以采用更少的溶液体积实现更高性能的涂层,体积的减少,可以大大降低促骨生长的昂贵的活性成分的用量,如生长因子等,从而在临床应用中大幅降低成本;
(3)制得的涂层为二水磷酸二钙(DCPD)和羟基磷灰石的复合物,具有更坚实的结构,并且沉积了更多的仿生CAP,能携带更多的活性蛋白,具有抗破骨吸收的耐酸性能,在酸性条件下释放的Ca2+更少,在体内降解更加缓慢,性能明显优于仅含有羟基磷灰石的涂层。
附图说明
图1为实施例4中钛片涂层厚度的检测结果示意图,其中(A)光镜下显微图像显示钛片在0.1、0.3、1、2、4和6mL的1×SCPS和4.5×SCPS溶液中分别孵育48h后横截面CaP结晶涂层;(B)定量分析SCPS溶液体积与数点计法测得的平均涂层厚度之间的关系;Bars=200μm。
图2为实施例5中在低放大倍数(1000×)和高放大倍数(5000×)下,分别观察4.5×SCPS和1×SCPS制备的仿生CaP涂层的形貌的场发射扫描电镜(FESEM)图像,其中Bar=20μm为低倍镜下图像、Bar=5μm为高倍镜下图像。
图3为实施例6中检测溶液的pH值,以及溶液中的Ca2+水平结果示意图,其中(A)为涂层制备过程中1×SCPS和4.5×SCPS溶液的pH值;(B)为Ca2+随时间的变化;*:与前一个时间点比较;p<0.05。
图4为实施例7中1×SCPS和4.5×SCPS涂层的EDS分析结果示意图,其中(A)为EDS能谱分析含无定形CaP涂层的钛片在1×SCPS或4.5×SCPS中孵育48h后表面CaP结晶涂层;(B)为利用EDS分析数据计算钙磷比。
图5为实施例7中的全反射-傅里叶变换红外光谱(ATR-FTIR)分析图谱,分析含无定形CaP涂层的钛片在1×SCPS(A)或4.5×SCPS(B)中孵育5h、24h和48h后CaP涂层的化学组成。
图6为实施例7中的x射线衍射(XRD)分析图谱,分析含无定形CaP涂层的钛片在1×SCPS(A)或4.5×SCPS(B)中孵育5h、24h和48h后CaP涂层的相组成。
图7为实施例8中在1×SCPS或4.5×SCPS中孵育获得的仿生CaP涂层钛片在中性(pH=7.4)和酸性(pH=6.0和4.5)缓冲液中Ca2+释放曲线图,其中,***:p<0.001。
图8为实施例9中模型蛋白异硫氰酸荧光素标记牛血清白蛋白(FITC-BSA)在1×SCPS或4.5×SCPS组钛片涂层中的包封率示意图,其中,***:p<0.001。
具体实施方式
下面结合附图和实施例对本发明作进一步详细描述,需要指出的是,以下所述实施例旨在便于对本发明的理解,而对其不起任何限定作用。
实施例1通过1×SCPS进行涂层
本实施例采用1×SCPS进行涂层,具体步骤为:
(1)分别配置5×SBF和1×SCPS溶液;
(2)取长和宽都是4mm,厚度为1mm的钛片,浸入5×SBF中,37℃孵育24h,以制备薄层无定形CaP(ACP)基底层。
(3)取出,干燥后,将含ACP涂层的钛片在37℃下浸泡在1×SCPS中,钛片浸没的溶液体积为1ml,孵育时间为24h。
获得1×SCPS涂层的钛片。
实施例2通过4.5×SCPS进行涂层
本实施例采用4.5×SCPS进行涂层,具体步骤为:
(1)分别配置5×SBF和4.5×SCPS溶液;
(2)取长和宽都是4mm,厚度为1mm的钛片,浸入5×SBF中,37℃孵育24h,以制备薄层无定形CaP(ACP)基底层。
(3)取出,干燥后,将含ACP涂层的钛片在室温下浸泡在4.5×SCPS中,钛片浸没的溶液体积为1ml,孵育时间为24h。
获得4.5×SCPS涂层的钛片。
实施例3通过5×SCPS进行涂层
本实施例采用5×SCPS进行涂层,具体步骤为:
(1)分别配置5×SBF和5×SCPS溶液;在配制SCPS溶液的过程中发现,随着SCPS浓度的增加,pH值必须降低,才能使其保持稳定的饱和状态并且不立即出现沉淀,配制5×SCPS溶液时,pH值需要在5.9左右才能保持稳定的饱和状态并且不立即出现沉淀。
(2)取长和宽都是4mm,厚度为1mm的钛片,浸入5×SBF中,37℃孵育24h,以制备薄层无定形CaP(ACP)基底层。
(3)取出,干燥后,将含ACP涂层的钛片浸泡在5×SCPS中,钛片浸没的溶液体积为1ml,孵育时间为24h。
研究发现,步骤(3)中,如果将含ACP涂层的钛片于37℃浸泡在5×SCPS中,5×SCPS溶液中快速出现沉淀,而钛片上并没有形成肉眼可见的结晶涂层,这主要是因为温度高,水分蒸发加快,离子运动加快,可见温度高时,高浓度的溶液体系不稳定。如果将含ACP涂层的钛片于室温下浸泡在5×SCPS中,5×SCPS溶液中虽然没有出现明显的快速沉淀,但钛片上依然没有形成结晶涂层。可见,无论在37℃或室温下,采用5×SCPS都无法形成结晶涂层。
5×SCPS无法形成结晶涂层的原因,可能是由于5×SCPS溶液的酸性导致无定形CaP基底层在溶液中迅速溶解。在5×SBF溶液中,由于CO2从溶液中释放,无定形CaP涂层始于pH值约为6、止于pH值高于8.2,这个pH远高于5.9。
与实施例2中使用4.5×SCPS制备涂层的情况进行比较,采用可以4.5×SCPS可以在无定形CaP涂层的钛片上重复制备出肉眼可见的结晶涂层,效果明显优于5×SCPS。因此,5×SCPS无法形成结晶涂层,建议优选采用4.5×SCPS进行涂层。
实施例4 4.5×SCPS和1×SCPS涂层制备厚度比较
本实施例中的1×SCPS涂层采用按照实施例1提供的方法制备的涂层,其中钛片浸没的1×SCPS溶液体积分别为0.1、0.3、1、2、4和6ml;4.5×SCPS涂层采用按照实施例2提供的方法制备的涂层,其中钛片浸没的4.5×SCPS溶液体积分别为0.1、0.3、1、2、4和6ml。
对1×SCPS涂层和4.5×SCPS涂层进行涂层厚度比较,考察在长、宽分别为4mm、厚度为1mm的钛片上形成的CaP结晶层的体积依赖性,涂层厚度的检测方法为:用甲基丙烯酸甲酯包埋钛片,沿钛片横截面垂直切出200μm厚样本并将其粘贴在有机玻璃支架上,研磨至80μm,进而用0.1%碱性品红染色、立体显微镜(Stemi SV6;Carl Zeiss,Jena,Germany)冷光源(KL 2500LCD;Carl Zeiss)观察并拍照。采用数点法测定涂层的表面积,涂层的平均厚度为涂层的表面积除以涂层钛表面的长度。涂层厚度的检测结果如图1所示,其中图1(A)光镜下显微图像显示钛片在0.1、0.3、1、2、4和6mL的1×SCPS和4.5×SCPS溶液中分别孵育48h后横截面CaP结晶涂层;图1(B)定量分析SCPS溶液体积与数点计法测得的平均涂层厚度之间的关系。Bars=200μm。
由图1可见,1×SCPS和4.5×SCPS组中,较高的SCPS体积产生较高的涂层厚度。0.3mL的4.5×SCPS组形成了连续的涂层,而0.3mL的1×SCPS形成了薄而不连续的涂层(图1A)。涂层是促进骨整合的一种方式,也是携带蛋白的方式,不连续的涂层,代表涂层不完整,促进骨整合能力降低,携带蛋白的量低。此外,1mL的4.5×SCPS已经能够形成非常厚的涂层(约110μm),而1mL的1×SCPS只能形成连续的薄涂层(29μm)。因此4.5×SCPS的涂层制备明显更厚。
图1(B)的定量数据表明,1×SCPS溶液体积与涂层厚度呈线性正相关(y=9.6957x+12.58,R2=0.9895),相关系数为0.99。该组的涂层厚度可以通过增加1×SCPS溶液的体积而成比例地增加。4.5×SCPS的体积与涂层的厚度呈双相关系。当4.5×SCPS的体积在0.1–1mL范围内时,涂层厚度快速增加;在较高的溶液体积(2–6mL)时,涂层厚度增加相对缓慢。4.5×SCPS体积与涂层厚度之间呈正对数相关关系(y=25.63ln(x)+92.109,R2=0.9895),相关系数约为0.99(图1B)。同时,由图1(B)可见,6mL 4.5×SCPS产生的涂层厚度为136±7μm,是6mL 1×SCPS(73±12μm)的1.9倍。可见,4.5×SCPS可以获得厚度更厚的涂层。
实施例5 4.5×SCPS和1×SCPS涂层的形貌表征比较
本实施例中的1×SCPS涂层采用按照实施例1提供的方法制备的涂层,其中钛片浸没的1×SCPS溶液体积分别为0.1、0.3、1、2、4和6ml,孵育时间为48小时;4.5×SCPS涂层采用按照实施例2提供的方法制备的涂层,其中钛片浸没的4.5×SCPS溶液体积分别为0.1、0.3、1、2、4和6ml,孵育时间为48小时。利用场发射扫描电子显微镜(FESEM,Phillips/FEIXL-30),在低放大倍数(1000×)和高放大倍数(5000×)下,分别观察4.5×SCPS和1×SCPS制备的仿生CaP涂层的形貌,场发射扫描电镜(FESEM)图像如图2所示,Bar=20μm为低倍镜下图像、Bar=5μm为高倍镜下图像。
由图2的左图可见,1×SCPS组中,1000×下,0.1mL 1×SCPS时,仍为球形或半球形形态,这是典型的无定形CaP,表示结晶层很薄;5000×下,0.1mL 1×SCPS时,观察到网状结构,类似于在较低体表面积比下获得的仿生涂层;当1×SCPS溶液体积增加到2mL时,也仍然可以看到无定形CaP形貌;1×SCPS在溶液体积为1mL时开始形成了典型的片状结晶层并且其尺寸随溶液体积的增加而增大。
无定型CaP降解很快,厚度很小,大概100-300纳米,携带蛋白量很少。本申请中的无定型CaP主要是作为前驱体,为后面的晶体形成提供结晶核。
由图2的右图可见,4.5×SCPS组中,即使在0.1mL 4.5×SCPS中也观察不到无定形CaP,但是晶体结构清晰可见。1ml 4.5×SCPS孵育后,片状结晶层显著增大并呈玫瑰状聚集。后续即使将4.5×SCPS的体积增加到6mL,涂层的形貌也没有进一步变化,晶体结构非常稳定。
实施例6 4.5×SCPS和1×SCPS涂层制备过程中溶液pH值和Ca2+浓度的变化比较
本实施例根据实施例6提供的基于体积相关涂层厚度分析,1.5mL 4.5×SCPS的涂层厚度(73μm)与6mL 1×SCPS在单一钛片(长和宽分别为4mm、厚度为1mm)上产生的涂层厚度相似。因此,本实施例中的1×SCPS涂层采用按照实施例1提供的方法制备的涂层,其中钛片浸没的1×SCPS溶液体积为6ml;4.5×SCPS涂层采用按照实施例2提供的方法制备的涂层,其中钛片浸没的4.5×SCPS溶液体积分别为1.5ml。
在孵育过程中,分别在不同时间点检测溶液的pH值,以及溶液中的Ca2+水平,使用原子吸收光谱仪(AAnalyst 100,PerkinElmer,Foster City,CA,USA)测量溶液中的Ca2+含量。数据以平均值和标准差表示,使用单因素方差分析(ANOVA)对有关pH值和Ca2+水平变化的数据进行比较,使用Bonferroni校正进行Post-hoc比较,显著性水平为p<0.05。
结果如图3所示,其中图3(A)为涂层制备过程中1×SCPS和4.5×SCPS溶液的pH值;图3(B)为Ca2+随时间的变化;*:与前一个时间点比较;p<0.05。
由图3(A)可见,与1×SCPS相比,4.5×SCPS溶液的初始pH值约为6.2。使用pH计,我们发现两种SCPS的pH值在涂层制备过程中都随着时间的推移而下降。对于1×SCPS,其pH值在前5小时内从7.4显著下降到大约7.0(p<0.05),并在后续阶段持续保持在这个水平;相比之下,4.5×SCPS的pH值在所有时间点(5、24、48h)均显著下降(p<0.05),最终下降至约5.75。
由图3(B)可见,与pH变化规律类似,1×SCPS组的Ca2+浓度在5h内显著下降,从4.0mM下降到3.2mM(p<0.05),并逐渐下降到2.6mM,涂层过程结束时约有35%的Ca2+被消耗;对于4.5×SCPS,在孵育5h和24h后,Ca2+浓度显著降低(p<0.05),但后续能稳定维持在约13mM,不再继续下降,涂层制备过程结束时约30%的Ca2+被消耗。
实施例7 4.5×SCPS和1×SCPS涂层的表征
本实施例中的1×SCPS涂层采用按照实施例1提供的方法制备的涂层,其中钛片浸没的1×SCPS溶液体积为6ml;4.5×SCPS涂层采用按照实施例2提供的方法制备的涂层,其中钛片浸没的4.5×SCPS溶液体积分别为1.5ml。通过EDS分析涂层中钙和磷的相对密度,计算钙磷比;利用XRD(X’Pert PRO;PANalytical,Malvern,UK)检测相组成、ATR-FTIR分析其化学基团。其中XRD分析的扫描范围为2.00°-80.00°、扫描速度为2°/min,扫描间隔为0.02°;ATR-FTIR检测的FTIR(Avatar 360Nicolet spectrometer;Thermo FisherScientific,Waltham,MA,USA)是从4000到400cm-1波数的透射光谱。
EDS分析结果如图4所示,其中,图4(A)为EDS能谱分析含无定形CaP涂层的钛片在1×SCPS或4.5×SCPS中孵育48h后表面CaP结晶涂层;图4(B)为利用EDS分析数据计算钙磷比。
由图4可见,1×SCPS和4.5×SCPS组钛片在孵育48h后形成的涂层主要由钙、磷和氧元素组成;涂层的Ca/P比值也表现出类似的变化规律:5h内先降低后升高,1×SCPS和4.5×SCPS的最终Ca/P比值分别为1.35和1.26。
图5为全反射-傅里叶变换红外光谱(ATR-FTIR)分析图谱,分析含无定形CaP涂层的钛片在1×SCPS(图5(A))或4.5×SCPS(图5(B))中孵育5h、24h和48h后CaP涂层的化学组成。
由图5可见,ATR-FTIR光谱显示1×SCPS和4.5×SCPS组的特征峰PO4 3-在波长1018-1021cm-1(v3)和560cm-1(v4),表明仿生CaP涂层成功地沉积到钛片。1×SCPS组钛片孵育5h后在1018-1021cm-1(v3)和560cm-1(v4)的峰更明显,其随着孵育时间的延长而更加显著(图5(A));相比之下,4.5×SCPS组钛片在孵育5h后的峰值明显比1×SCPS组更显著(图5(B))。随着时间的推移,特征峰变得更为显著,这表明5h的孵育即可形成明显的晶体涂层。
图6为x射线衍射(XRD)分析图谱,分析含无定形CaP涂层的钛片在1×SCPS(图6(A))或4.5×SCPS(图6(B))中孵育5h、24h和48h后CaP涂层的相组成。其中,2θ=35.5°、38.4°、40.3°、53.4°、63.1°和71.4°处的特征衍射峰对应于钛的(1000)、(0002)、(1011)、(1012)、(1120)和(1013)面。
由图6(A)可以看出,经1×SCPS孵育形成的涂层在2θ=26.1°和31.5°处有两个特征峰,分别对应于羟基磷灰石(JCPDS no.09-0432)的(002)和(211)面;在1×SCPS中孵育5h后,这些峰开始显现,随着涂层沉积过程的进行,这些峰变得更加明显。
同样,由图6(B)可以看出,在4.5×SCPS中形成的涂层也具有羟基磷灰石的两个特征峰;此外,该涂层在2θ=11.7°处还存在一个非常尖锐的高峰,对应于二水磷酸二钙(DCPD)(JCPDS no.9-77)。这些发现表明该涂层是DCPD和磷灰石的复合物。DCPD具有生物相容性、生物可降解性和骨传导性,可转化为无水磷酸二钙(DCPA)(pH<6)、磷酸八钙(OCP)(pH=6-7)或沉淀羟基磷灰石(pHAp)(pH>7)。在医学上,DCPD可用于CaP骨水泥的制备。研究发现,DCPD在体内既可以转化为羟基磷灰石,也可以降解并被骨取代。
实施例8 4.5×SCPS和1×SCPS涂层中Ca2+的释放曲线
本实施例中的1×SCPS涂层采用按照实施例1提供的方法制备的涂层,其中钛片浸没的1×SCPS溶液体积为6ml,孵育时间为48h;4.5×SCPS涂层采用按照实施例2提供的方法制备的涂层,其中钛片浸没的4.5×SCPS溶液体积分别为1.5ml,孵育时间为48h。
检测不同pH条件下涂层中Ca2+释放曲线的方法为:为了检测两组涂层中Ca2+释放动力学,将含涂层的钛片浸泡在0.5mL如下的缓冲液中:0.05M Tris-HCL(pH=7.424)或0.1M醋酸缓冲液(pH=6.0或4.529),60转/分钟、37℃孵育。在预定的4、5、9h、1、2、3、5、8、11、14、17、21、27、35天的时间点,收获上清并加入相同体积的新缓冲液。最后,将样品用镧试剂[0.5wt%La(NO3)3·6H2O(Merck,Darmstadt,Germany)]在0.05M HCl中稀释后,使用原子吸收光谱仪(AAnalyst 100,PerkinElmer,Foster City,CA,USA)测量溶液中的Ca2+含量,并分析累积释放曲线。数据以平均值和标准差表示。使用单因素方差分析(ANOVA)对有关pH值和Ca2+水平变化的数据进行比较,使用Bonferroni校正进行Post-hoc比较,使用非配对t检验来比较同一缓冲液中释放的Ca2+总量,显著性水平为p<0.05。
在1×SCPS或4.5×SCPS中孵育获得的仿生CaP涂层钛片在中性(pH=7.4)和酸性(pH=6.0和4.5)缓冲液中Ca2+释放曲线如图7所示,其中,***:p<0.001。
由图7可见,选择酸性溶液来模拟手术部位(初始环境可能是酸性的)以及破骨细胞(通过酸分泌降解骨)的作用,在生理条件下,pH为7.4时,Ca2+的释放量较低。两种SCPSs中Ca2+的释放曲线相同,平均每天释放约0.17μg。在pH为6.0的缓冲液中,两种涂层的释放谱仍基本一致,平均每天释放量均为0.78μg。这一现象与锌离子从羟基磷灰石涂层中溶解曲线一致,锌离子在乙酸缓冲液(pH=4.5)中释放速率较高,在磷酸盐缓冲盐水中较低。在强酸性条件下(pH=4.5),1×SCPS组释放的Ca2+总量(59.95±1.39μg)显著高于4.5×SCPS组(32.67±0.03μg)(p<0.001)。而即使在强酸性微环境中,4.5×SCPS涂层中Ca2+释放没有明显变化,这可以解释为其坚实的结构和有更多的仿生CaP沉积在钛片上。1×SCPS组Ca2+溶解速度较快是因为游离的H+影响了结构疏松磷灰石的成核。这一发现表明,在酸性条件(pH=4.5),4.5×SCPS组涂层释放的Ca2+明显少于1×SCPS组,可见用4.5×SCPS制备的仿生CaP涂层具有抗破骨吸收的耐酸性能,显示其在体内可能降解缓慢。
实施例9 4.5×SCPS和1×SCPS涂层对模型蛋白FITC-BSA的包封率比较
根据实施例6提供的体积依赖性涂层厚度分析显示,1.5mL 4.5×SCPS产生的涂层厚度(约73μm)与6mL 1×SCPS在单一钛片(长、宽分别为4mm,厚度为1mm)上产生的涂层厚度相似。因此,本实施例中的1×SCPS涂层采用按照实施例1提供的方法制备的涂层,其中钛片浸没的1×SCPS溶液体积为6ml;4.5×SCPS涂层采用按照实施例2提供的方法制备的涂层,其中钛片浸没的4.5×SCPS溶液体积分别为1.5ml。
基于前期研究,模型蛋白异硫氰酸荧光素标记牛血清白蛋白FITC-BSA(5μg/mL)为模型蛋白。将FITC-BSA以5μg/mL的浓度分别溶于1×SCPS或4.5×SCPS中,再分别孵育时间为48h。包被后,将样本涂层溶解于0.5mL 0.5M EDTA(pH=8.0)中,涡旋两次各5分钟以回收包被的FITC-BSA。用分光光度计(激发波长:485nm、发射波长:519nm)读取荧光密度并绘制FITC-BSA标准曲线以计算FITC-BSA的含量。包封率:包埋的FITC-BSA/添加到SCPS中的FITC-BSA(n=5)。图8为模型蛋白异硫氰酸荧光素标记牛血清白蛋白(FITC-BSA)在1×SCPS或4.5×SCPS组钛片涂层中的包封率示意图,其中,***:p<0.001。
由图8可见,FITC-BSA在4.5×SCPS涂层中的包封率为81.20±6.42%,显著高于1×SCPS涂层的21.86±1.90%(p<0.001)。4.5×SCPS组之所以有如此高FITC-BSA的包封率,很大程度上是由于溶液体积的减少和涂层产量的提升。
虽然本发明披露如上,但本发明并非限定于此。任何本领域技术人员,在不脱离本发明的精神和范围内,均可作各种更动与修改,因此本发明的保护范围应当以权利要求所限定的范围为准。
Claims (6)
1.一种双相磷酸钙涂层,其特征在于,所述涂层含有二水磷酸二钙和羟基磷灰石;所述涂层的制备方法由以下步骤组成:
(1)将钛片浸入5×SBF中,37℃孵育24h,制备CaP基底层;
(2)取出,干燥后,浸入4.5×SCPS中,室温下孵育5h以上。
2.如权利要求1所述的双相磷酸钙涂层的制备方法,其特征在于,由以下步骤组成:
(1)将钛片浸入5×SBF中,37℃孵育24h,制备CaP基底层;
(2)取出,干燥后,浸入4.5×SCPS中,室温下孵育5h以上。
3.如权利要求2所述的制备方法,其特征在于,其中,5×SBF的无机离子组成为:Na+ 715.0 mM,Ca2+ 12.5 mM,Mg2+ 7.5 mM,Cl- 724.0 mM,HPO4 2- 5.0 mM,HCO3 - 21.0 mM,pH6.0,温度37℃;4.5×SCPS的无机离子组成为:Na+ 630.0 mM,Ca2+ 18.0 mM,Cl- 828.0 mM,HPO4 2- 9.0 mM,pH6.2,室温。
4.如权利要求3所述的制备方法,其特征在于,所述钛片的长和宽都是4mm,厚度为1mm。
5.如权利要求4所述的制备方法,其特征在于,步骤(2)所述的4.5×SCPS的体积为0.1-6ml。
6.4.5×SCPS在用于制备如权利要求1所述的双相磷酸钙涂层方面的用途。
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