技术领域Technical Field
本发明涉及医用材料领域技术领域，具体涉及一种氧化锌/磷酸锌纳米棒复合抗菌涂层及其制备方法和应用。The invention relates to the technical field of medical materials, and in particular to a zinc oxide/zinc phosphate nanorod composite antibacterial coating and a preparation method and application thereof.
背景技术Background technique
随着生物材料产业的发展，临床上医用材料在人体组织损伤修复方面有着广泛的应用。例如生物医用金属材料钛及钛合金已经广泛应用于人工关节、牙科植入体、骨科假体等领域；生物医用高分子材料聚醚醚酮也在椎间融合器、颅面损伤修复方面有着广泛的应用。然而在实际临床应用中，医用材料仍面对着细菌感染这个亟需解决的问题。术后感染的发生在很大程度上影响着手术的效果，而某些植入体术后感染往往是灾难性的。例如人工关节置换手术的植入体术后感染需要二次手术清创处理，给患者带来严重的生理、精神和经济负担。术后感染一般是由粘附并定殖于医用材料表面的细菌引起的。因此在医用材料表面制备抗菌涂层，能够极大程度上减少术后感染的发生。另外，在材料表面引入抗菌涂层时需要同时对其生物学毒性进行考量，使抗菌涂层具有优异抗感染性能的同时不能延缓甚至阻碍组织的修复进程。With the development of the biomaterials industry, medical materials have been widely used in the repair of human tissue damage in clinical practice. For example, biomedical metal materials titanium and titanium alloys have been widely used in artificial joints, dental implants, orthopedic prostheses and other fields; biomedical polymer materials polyetheretherketone are also widely used in intervertebral fusion and craniofacial injury repair. However, in actual clinical applications, medical materials still face the urgent problem of bacterial infection. The occurrence of postoperative infection greatly affects the effect of surgery, and postoperative infection of some implants is often catastrophic. For example, postoperative infection of implants in artificial joint replacement surgery requires secondary surgical debridement, which brings serious physiological, mental and economic burdens to patients. Postoperative infection is generally caused by bacteria that adhere to and colonize on the surface of medical materials. Therefore, preparing an antibacterial coating on the surface of medical materials can greatly reduce the occurrence of postoperative infection. In addition, when introducing an antibacterial coating on the surface of the material, its biological toxicity needs to be considered at the same time, so that the antibacterial coating has excellent anti-infection properties while not delaying or even hindering the repair process of the tissue.
因此，在医用材料表面制备同时兼具抗感染和促组织愈合能力的涂层对于医用材料领域具有重要意义。Therefore, preparing coatings on the surface of medical materials that have both anti-infection and tissue healing capabilities is of great significance in the field of medical materials.
发明内容Summary of the invention
为解决上述技术问题，本发明提供一种氧化锌/磷酸锌纳米棒复合抗菌涂层及其制备方法和应用。In order to solve the above technical problems, the present invention provides a zinc oxide/zinc phosphate nanorod composite antibacterial coating and a preparation method and application thereof.
为实现上述目的，本发明采用的技术方案如下：To achieve the above purpose, the technical solution adopted by the present invention is as follows:
本发明第一方面提供一种氧化锌/磷酸锌纳米棒复合抗菌涂层的制备方法，该方法包括先在医用材料表面制备氧化锌纳米棒涂层；再通过在磷酸盐或磷酸氢盐溶液中水浴处理的方法将所述氧化锌纳米棒涂层中的氧化锌转换成磷酸锌，从而制得具有纳米形貌的氧化锌/磷酸锌纳米棒复合抗菌涂层。The first aspect of the present invention provides a method for preparing a zinc oxide/zinc phosphate nanorod composite antibacterial coating, which comprises first preparing a zinc oxide nanorod coating on the surface of a medical material; and then converting the zinc oxide in the zinc oxide nanorod coating into zinc phosphate by a water bath treatment in a phosphate or hydrogen phosphate solution, thereby preparing a zinc oxide/zinc phosphate nanorod composite antibacterial coating with a nano-morphology.
优选的，该方法具体包括以下步骤：步骤一、在医用材料表面制备氧化锌种子层，然后通过水热处理在所述氧化锌种子层上形成氧化锌纳米棒涂层，获得负载有氧化锌纳米棒涂层的医用材料；步骤二、将所述负载有氧化锌纳米棒涂层的医用材料浸入至磷酸盐或磷酸氢盐溶液中进行磷酸化处理，通过调控反应条件控制氧化锌向磷酸锌的转换比例，获得氧化锌/磷酸锌纳米棒复合抗菌涂层。Preferably, the method specifically comprises the following steps: step 1, preparing a zinc oxide seed layer on the surface of the medical material, and then forming a zinc oxide nanorod coating on the zinc oxide seed layer by hydrothermal treatment to obtain a medical material loaded with the zinc oxide nanorod coating; step 2, immersing the medical material loaded with the zinc oxide nanorod coating in a phosphate or hydrogen phosphate solution for phosphorylation, and controlling the conversion ratio of zinc oxide to zinc phosphate by adjusting the reaction conditions to obtain a zinc oxide/zinc phosphate nanorod composite antibacterial coating.
优选的，所述步骤二的反应条件为：控制反应体系的pH值为8.0-10.0，温度为20℃-100℃，反应时间为1h-24h。Preferably, the reaction conditions of step 2 are: controlling the pH value of the reaction system to 8.0-10.0, the temperature to 20° C.-100° C., and the reaction time to 1 h-24 h.
优选的，所述步骤二的反应条件为：控制反应体系的pH值为8.0，温度为60℃，反应时间为2h。Preferably, the reaction conditions of step 2 are: controlling the pH value of the reaction system to 8.0, the temperature to 60° C., and the reaction time to 2 h.
优选的，所述磷酸盐或磷酸氢盐溶液为磷酸钾、磷酸钠、磷酸氢二钾、磷酸氢二钠、磷酸二氢钾或磷酸二氢钠中的一种或多种混合。即，所述磷酸盐或磷酸氢盐溶液包括且不限于钾盐，也可以是其他金属元素的磷酸盐或磷酸氢盐。Preferably, the phosphate or hydrogen phosphate solution is a mixture of one or more of potassium phosphate, sodium phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate or sodium dihydrogen phosphate. That is, the phosphate or hydrogen phosphate solution includes but is not limited to potassium salt, and may also be phosphates or hydrogen phosphates of other metal elements.
优选的，所述磷酸盐或磷酸氢盐溶液的浓度为0.01mol/L-0.5mol/L。Preferably, the concentration of the phosphate or hydrogen phosphate solution is 0.01 mol/L-0.5 mol/L.
优选的，所述磷酸盐或磷酸氢盐溶液的浓度为0.1mol/L。Preferably, the concentration of the phosphate or hydrogen phosphate solution is 0.1 mol/L.
优选的，所述步骤一具体包括以下步骤，配制混合溶液：将醋酸锌(Zn(CH3COO)2·2H2O)与乙醇胺(ethanolamine)溶解于乙醇中，并且室温搅拌5h，形成混合溶液；其中，所述醋酸锌、乙醇胺和乙醇的浓度均为0.05mol/L；进行旋涂处理：将样品固定于旋涂仪，并取60μl以上所述混合溶液滴加于所述样品的表面后，将所述样品至于烘箱中于120℃处理10min；制备形成氧化锌种子层的样品：将所述旋涂处理重复三次后，将所述样品转移至马弗炉中于500℃处理30min，从而在样品表面形成氧化锌种子层；制备氧化锌纳米棒涂层：将形成氧化锌种子层的样品放入水热釜中，在含有0.025mol/L硝酸锌(Zn(NO3)2·6H2O)和0.025mol/L六亚甲基四胺(hexamethylenetetramine)的水溶液中于90℃处理5h，获得所述氧化锌纳米棒涂层。其中，所述的样品为医用材料、医用植入材料。Preferably, the step 1 specifically includes the following steps: preparing a mixed solution: dissolving zinc acetate (Zn(CH3 COO)2 ·2H2 O) and ethanolamine in ethanol, and stirring at room temperature for 5 hours to form a mixed solution; wherein the concentrations of the zinc acetate, ethanolamine and ethanol are all 0.05 mol/L; performing a spin coating process: fixing the sample on a spin coater, taking 60 μl of the mixed solution and dripping it on the surface of the sample, and then placing the sample in an oven and treating it at 120° C. for 10 minutes; preparing a sample to form a zinc oxide seed layer: after repeating the spin coating process three times, transferring the sample to a muffle furnace and treating it at 500° C. for 30 minutes, thereby forming a zinc oxide seed layer on the surface of the sample; preparing a zinc oxide nanorod coating: placing the sample to form a zinc oxide seed layer in a hydrothermal reactor, and in a solution containing 0.025 mol/L zinc nitrate (Zn(NO3 )2 ·6H2 The zinc oxide nanorod coating is obtained by treating the zinc oxide nanorod coating in an aqueous solution of 0.025 mol/L hexamethylenetetramine at 90° C. for 5 hours. The sample is a medical material or a medical implant material.
本发明第二方面提供一种氧化锌/磷酸锌纳米棒复合抗菌涂层，如上述的氧化锌/磷酸锌纳米棒复合抗菌涂层的制备方法所制得的氧化锌/磷酸锌纳米棒复合抗菌涂层。The second aspect of the present invention provides a zinc oxide/zinc phosphate nanorod composite antibacterial coating, such as the zinc oxide/zinc phosphate nanorod composite antibacterial coating prepared by the above-mentioned method for preparing the zinc oxide/zinc phosphate nanorod composite antibacterial coating.
本发明第三方面也提供如上述的氧化锌/磷酸锌纳米棒复合抗菌涂层在医用材料领域的应用，尤其适用于医用植入材料领域。The third aspect of the present invention also provides the use of the zinc oxide/zinc phosphate nanorod composite antibacterial coating in the field of medical materials, especially in the field of medical implant materials.
相较于现有技术，本发明提供的技术方案至少具有以下优点：Compared with the prior art, the technical solution provided by the present invention has at least the following advantages:
本发明提供一种氧化锌/磷酸锌纳米棒复合抗菌涂层及其制备方法和应用。该制备方法首先在医用材料表面制备氧化锌纳米棒涂层，然后在磷酸盐或磷酸氢盐溶液中水浴处理将氧化锌纳米棒涂层中的氧化锌部分转换成降解速率更低的磷酸锌。通过调控水浴处理的参数可以调控磷酸锌的转换比例，从而调控涂层的整体降解速率。该涂层可以均匀、高效地制备于形状复杂的医用材料表面，其工艺过程简单、成本低廉、适用于批量及工业化生产。The present invention provides a zinc oxide/zinc phosphate nanorod composite antibacterial coating and a preparation method and application thereof. The preparation method first prepares a zinc oxide nanorod coating on the surface of a medical material, and then performs a water bath treatment in a phosphate or hydrogen phosphate solution to convert the zinc oxide part in the zinc oxide nanorod coating into zinc phosphate with a lower degradation rate. The conversion ratio of zinc phosphate can be adjusted by adjusting the parameters of the water bath treatment, thereby adjusting the overall degradation rate of the coating. The coating can be uniformly and efficiently prepared on the surface of a medical material with a complex shape, and the process is simple, low-cost, and suitable for batch and industrial production.
制得的具有纳米形貌的氧化锌/磷酸锌复合涂层不但具有良好的抗感染能力，而且大大降低了原有氧化锌纳米棒涂层的生物学毒性。其中，通过控制合适的磷酸锌转换比例，可以达到在降解过程中释放的适量锌离子，从而实现促进组织愈合的目的。The prepared zinc oxide/zinc phosphate composite coating with nano-morphology not only has good anti-infection ability, but also greatly reduces the biological toxicity of the original zinc oxide nanorod coating. Among them, by controlling the appropriate zinc phosphate conversion ratio, the appropriate amount of zinc ions released during the degradation process can be achieved, thereby achieving the purpose of promoting tissue healing.
将该氧化锌/磷酸锌复合涂层作为抗菌涂层应用在医用领域，可以获得兼具抗感染和促愈合双重功能的医用材料，可以减少植入后感染情况的发生，特别是骨科硬组织植入材料表面，例如金属基的钛及钛合金、医用不锈钢等。与此同时，该涂层赋予医用材料表面抗感染能力的同时，没有生物学毒性，并且可以促进组织修复。The zinc oxide/zinc phosphate composite coating is used as an antibacterial coating in the medical field to obtain medical materials with dual functions of anti-infection and healing promotion, which can reduce the occurrence of post-implantation infection, especially on the surface of orthopedic hard tissue implant materials, such as metal-based titanium and titanium alloys, medical stainless steel, etc. At the same time, the coating gives the surface of medical materials anti-infection ability without biological toxicity and can promote tissue repair.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
一个或多个实施例通过与之对应的附图中的图片进行示例性说明，这些示例性说明并不构成对实施例的限定，除非有特别申明，附图中的图不构成比例限制。One or more embodiments are exemplarily described by pictures in the corresponding drawings, and these exemplifications do not constitute limitations on the embodiments. Unless otherwise stated, the pictures in the drawings do not constitute proportional limitations.
图1a是实施例1中Ti-ZnO样品表面的扫描电子显微镜照片；FIG1a is a scanning electron microscope photograph of the surface of the Ti-ZnO sample in Example 1;
图1b是实施例1中Ti-ZnP0.5样品表面的扫描电子显微镜照片；FIG1b is a scanning electron microscope photograph of the surface of the Ti-ZnP0.5 sample in Example 1;
图1c是实施例1中Ti-ZnP1样品表面的扫描电子显微镜照片；FIG1c is a scanning electron microscope photograph of the surface of the Ti-ZnP1 sample in Example 1;
图1d是实施例1中Ti-ZnP2样品表面的扫描电子显微镜照片；FIG1d is a scanning electron microscope photograph of the surface of the Ti-ZnP2 sample in Example 1;
图1e是实施例1中Ti-ZnP3样品表面的扫描电子显微镜照片；FIG1e is a scanning electron microscope photograph of the surface of the Ti-ZnP3 sample in Example 1;
图2是实施例2中将Ti-ZnO样品在含有0.1M磷酸氢二钾(K2HPO4·3H2O)的水溶液中于60℃、70℃和80℃处理2h后所获得样品的扫描电子显微镜照片；FIG2 is a scanning electron microscope photograph of a sample obtained after treating a Ti-ZnO sample in an aqueous solution containing 0.1M potassium dihydrogen phosphate (K2 HPO4 ·3H2 O) at 60° C., 70° C. and 80° C. for 2 h in Example 2;
图3a是实施例3中Ti-ZnO样品表面的透射电子显微镜照片；FIG3a is a transmission electron microscope photograph of the surface of the Ti-ZnO sample in Example 3;
图3b是实施例3中Ti-ZnP1样品表面的透射电子显微镜照片；FIG3 b is a transmission electron microscope photograph of the surface of the Ti-ZnP1 sample in Example 3;
图3c是实施例3中Ti-ZnP2样品表面的透射电子显微镜照片；FIG3c is a transmission electron microscope photograph of the surface of the Ti-ZnP2 sample in Example 3;
图4a是实施例4中各组样品的X射线衍射分析；FIG4a is an X-ray diffraction analysis of each group of samples in Example 4;
图4b是实施例4中各组样品的X射线光电子能谱全谱谱图；FIG4 b is a full spectrum of X-ray photoelectron spectroscopy of each group of samples in Example 4;
图4c是实施例4中各组处理样品表面元素的原子百分比；FIG4c is the atomic percentage of the surface elements of each group of treated samples in Example 4;
图4d是实施例4中各组处理样品表面的亲疏水性；FIG4d is the hydrophilicity and hydrophobicity of the surface of each group of treated samples in Example 4;
图5是实施例5中各组样品表面经过划痕实验后的扫描电子显微镜照片；FIG5 is a scanning electron microscope photograph of the surfaces of each group of samples in Example 5 after a scratch test;
图6是实施例6中各组样品在Hank’s缓冲液中浸泡30天后锌离子的释放趋势；Fig. 6 is the release trend of zinc ions after each group of samples in Example 6 were immersed in Hank's buffer for 30 days;
图7是实施例7中各组样品在Hank’s缓冲液中浸泡30中各时间点样品表面的扫描电子显微镜照片；FIG7 is a scanning electron microscope photograph of the sample surface of each group of samples in Example 7 at various time points after being immersed in Hank's buffer for 30 minutes;
图8是实施例8中骨髓间充质干细胞在各组样品表面培养后的增殖情况；FIG8 is a diagram showing the proliferation of bone marrow mesenchymal stem cells after culture on the surface of each group of samples in Example 8;
图9a是实施例9中骨髓间充质干细胞在各组样品表面向成骨细胞诱导分化后碱性磷酸酶的活性；FIG9a is the alkaline phosphatase activity of bone marrow mesenchymal stem cells after they were induced to differentiate into osteoblasts on the surface of each group of samples in Example 9;
图9b是实施例9中骨髓间充质干细胞在各组样品表面向成骨细胞诱导分化后细胞的胶原分泌情况；FIG9b is a diagram showing the collagen secretion of bone marrow mesenchymal stem cells after they were induced to differentiate into osteoblasts on the surface of each group of samples in Example 9;
图9c是实施例9中骨髓间充质干细胞在各组样品表面向成骨细胞诱导分化后细胞外基质的矿化情况；FIG9c is a diagram showing the mineralization of the extracellular matrix after bone marrow mesenchymal stem cells were induced to differentiate into osteoblasts on the surface of each group of samples in Example 9;
图10a是实施例10中通过平板涂布法检测在各组样品表面培养的金黄色葡萄球菌和大肠杆菌的存活情况照片；FIG10a is a photograph showing the survival of Staphylococcus aureus and Escherichia coli cultured on the surface of each group of samples detected by the plate coating method in Example 10;
图10b是实施例10中计算得到的各组样品表面对于金黄色葡萄球菌和大肠杆菌的抗菌率。FIG. 10 b shows the antibacterial rates of the surfaces of each group of samples against Staphylococcus aureus and Escherichia coli calculated in Example 10.
具体实施方式Detailed ways
为了解决现有技术中存在的问题，发明人发现氧化锌纳米棒涂层具有制备工艺简单、成本低廉、适合制备在大面积复杂表面等优势。更重要的是，氧化锌纳米棒涂层具有广谱抗菌活性，有望在医用材料领域作为抗菌涂层得到广泛应用。一般认为，氧化锌纳米棒阵列的广谱抗菌性能来自于三方面：1)涂层降解释放的锌离子的杀菌作用；2)氧化锌本身作为半导体材料产生的活性氧自由基(ROS)对于细菌的细胞膜、细胞质以及遗传物质的破坏作用；3)纳米棒形貌对细菌的细胞壁的物理穿刺作用。然而氧化锌纳米棒涂层在生理条件下降解速率过快，降解过程中突释出大量的锌离子，并且与氧化锌产生的ROS的综合作用下对组织细胞也会产生较强的毒性。因此，为改善氧化锌纳米棒涂层的抗菌应用前景，有必要对其降解速率进行调节，从而在维持其良好的抗菌性能的前提下使其同样具有良好的促组织修复功能。In order to solve the problems existing in the prior art, the inventors found that the zinc oxide nanorod coating has the advantages of simple preparation process, low cost, and suitable for preparation on large-area complex surfaces. More importantly, the zinc oxide nanorod coating has broad-spectrum antibacterial activity and is expected to be widely used as an antibacterial coating in the field of medical materials. It is generally believed that the broad-spectrum antibacterial performance of the zinc oxide nanorod array comes from three aspects: 1) the bactericidal effect of zinc ions released by coating degradation; 2) the destructive effect of reactive oxygen free radicals (ROS) generated by zinc oxide itself as a semiconductor material on the cell membrane, cytoplasm and genetic material of bacteria; 3) the physical puncture effect of the nanorod morphology on the cell wall of bacteria. However, the degradation rate of the zinc oxide nanorod coating is too fast under physiological conditions, and a large amount of zinc ions are suddenly released during the degradation process, and the combined effect of the ROS generated by zinc oxide will also produce strong toxicity to tissue cells. Therefore, in order to improve the antibacterial application prospects of the zinc oxide nanorod coating, it is necessary to adjust its degradation rate so that it can also have a good tissue repair function while maintaining its good antibacterial performance.
发明人经过研究发现，通过对材料表面制备的氧化锌纳米棒涂层进行磷酸化处理，将其部分转换成磷酸锌，可以在维持其纳米棒状形貌和优异抗菌性能的前提下，降低涂层的降解速率。合适的降解速率下释放出的锌离子不但不会引发生物学毒性，而且能够促进组织细胞的修复功能。The inventors have found that by phosphorylating the zinc oxide nanorod coating prepared on the surface of the material and converting it partially into zinc phosphate, the degradation rate of the coating can be reduced while maintaining its nanorod morphology and excellent antibacterial properties. The zinc ions released at a suitable degradation rate will not only not cause biological toxicity, but also promote the repair function of tissue cells.
基于此，本发明提供一种在医用材料表面制备具有纳米棒形貌的氧化锌/磷酸锌复合涂层的方法。该涂层既拥有良好的抗感染性能，又不会产生生物学毒性并且能够促进组织修复。Based on this, the present invention provides a method for preparing a zinc oxide/zinc phosphate composite coating with nanorod morphology on the surface of a medical material. The coating has good anti-infection performance, does not produce biological toxicity, and can promote tissue repair.
下面结合具体实施方式对本发明进行详细说明。The present invention is described in detail below in conjunction with specific implementation modes.
下面以金属基医用骨科植入材料纯钛作为基底，在其表面构建氧化锌/磷酸锌纳米棒复合涂层来证明本发明的可行性。The feasibility of the present invention is demonstrated by using pure titanium, a metal-based medical orthopedic implant material, as a substrate and constructing a zinc oxide/zinc phosphate nanorod composite coating on its surface.
实施例1Example 1
将直径14mm、厚2mm的医用级纯钛片材经2000目砂纸打磨后，依次用丙酮、酒精、去离子水超声清洗干净。该预处理后样品标记为Ti。A medical grade pure titanium sheet with a diameter of 14 mm and a thickness of 2 mm was polished with 2000-grit sandpaper and then ultrasonically cleaned with acetone, alcohol, and deionized water in sequence. The pretreated sample was marked as Ti.
将醋酸锌(Zn(CH3COO)2·2H2O)与乙醇胺(ethanolamine)溶解于乙醇中，使其浓度都为0.05M并且室温搅拌5小时。将Ti固定于旋涂仪并取60μl以上溶液滴加于Ti表面后，将样品至于烘箱中于120℃处理10min。以上处理重复三次后，样品转移至马弗炉中于500℃处理30min，从而在Ti表面形成ZnO“种子层”。然后将样品放入水热釜中，在含有0.025M硝酸锌(Zn(NO3)2·6H2O)和0.025M六亚甲基四胺(hexamethylenetetramine)的水溶液中于90℃处理5h，从而获得氧化锌纳米棒涂层。所获得样品标记为Ti-ZnO。其中，“M”为“mol/L”。Zinc acetate (Zn(CH3 COO)2 ·2H2 O) and ethanolamine were dissolved in ethanol to a concentration of 0.05 M and stirred at room temperature for 5 hours. Ti was fixed on a spin coater and 60 μl of the above solution was dripped onto the Ti surface. The sample was placed in an oven and treated at 120°C for 10 min. After the above treatment was repeated three times, the sample was transferred to a muffle furnace and treated at 500°C for 30 min to form a ZnO "seed layer" on the Ti surface. The sample was then placed in a hydrothermal autoclave and treated at 90°C for 5 h in an aqueous solution containing 0.025 M zinc nitrate (Zn(NO3 )2 ·6H2 O) and 0.025 M hexamethylenetetramine to obtain a zinc oxide nanorod coating. The obtained sample is labeled Ti-ZnO. Wherein, "M" is "mol/L".
进一步将Ti-ZnO在含有0.1M磷酸氢二钾(K2HPO4·3H2O)的水溶液中于60℃处理若干小时后，可将氧化锌纳米棒阵列部分转换成磷酸锌。所获得样品分别标记为Ti-ZnP0.5、Ti-ZnP1、Ti-ZnP2、和Ti-ZnP3，其中数字表示处理的小时数。After further treating Ti-ZnO in an aqueous solution containing0.1M potassium dihydrogen phosphate (K2HPO4 ·3H2O ) at 60°C for several hours, the zinc oxide nanorod arrays can be partially converted into zinc phosphate. The obtained samples are marked as Ti-ZnP0.5, Ti-ZnP1, Ti-ZnP2, and Ti-ZnP3, where the numbers represent the number of hours of treatment.
使用扫描电子显微镜(SEM)观察不同样品的表面形貌。图1a-e分别为氧化锌纳米棒涂层、Ti-ZnP0.5、Ti-ZnP1、Ti-ZnP2、和Ti-ZnP3的SEM图片。由图可见当反应0.5h时，表面形貌相比于氧化锌纳米棒涂层基本没有变化。反应1h后纳米棒底部开始转化成块状，而此变化在2h和3h后更加明显。The surface morphology of different samples was observed using a scanning electron microscope (SEM). Figures 1a-e are SEM images of zinc oxide nanorod coating, Ti-ZnP0.5, Ti-ZnP1, Ti-ZnP2, and Ti-ZnP3, respectively. It can be seen from the figure that when the reaction is 0.5h, the surface morphology is basically unchanged compared to the zinc oxide nanorod coating. After 1h of reaction, the bottom of the nanorod begins to transform into a block, and this change is more obvious after 2h and 3h.
实施例2Example 2
将实施例1中的Ti-ZnO在含有0.1M磷酸氢二钾(K2HPO4·3H2O)的水溶液中于60℃、70℃和80℃处理2h后，使用扫描电子显微镜(SEM)观察不同样品的表面形貌(图2)。由图可见随着温度的升高，纳米棒底部转换成块状的趋势更加明显，说明温度的升高更有利于氧化锌向磷酸锌的转换。After the Ti-ZnO in Example 1 was treated in an aqueous solution containing 0.1M potassium dihydrogen phosphate (K2 HPO4 ·3H2 O) at 60°C, 70°C and 80°C for 2 hours, the surface morphology of different samples was observed using a scanning electron microscope (SEM) ( FIG. 2 ). As can be seen from the figure, as the temperature increases, the trend of the bottom of the nanorods converting into a block becomes more obvious, indicating that the increase in temperature is more conducive to the conversion of zinc oxide to zinc phosphate.
实施例3Example 3
使用透射电子显微镜(TEM)观察各组样品表面。图3a-c分别为氧化锌纳米棒涂层、Ti-ZnP1和Ti-ZnP2的TEM图片。由图3a可见，1.92nm的晶面间距对应于氧化锌晶体。而在反应1h的样品(图3b)和2h的样品(图3c)中，可以同时观察到氧化锌和磷酸锌的晶体结构。说明在经过在磷酸氢盐溶液中处理后，氧化锌纳米棒涂层中的部分氧化锌转换成了磷酸锌。The surface of each group of samples was observed using a transmission electron microscope (TEM). Figures 3a-c are TEM images of zinc oxide nanorod coating, Ti-ZnP1 and Ti-ZnP2, respectively. As shown in Figure 3a, the interplanar spacing of 1.92 nm corresponds to zinc oxide crystals. In the sample with a reaction time of 1 h (Figure 3b) and 2 h (Figure 3c), the crystal structures of zinc oxide and zinc phosphate can be observed at the same time. This indicates that after being treated in a hydrogen phosphate solution, part of the zinc oxide in the zinc oxide nanorod coating is converted into zinc phosphate.
实施例4Example 4
使用X射线衍射(XRD)分析涂层的晶体结构。由图4a可知Ti-ZnP1和Ti-ZnP2样品表面的涂层同时存在着氧化锌和磷酸锌两种晶型。The crystal structure of the coating was analyzed by X-ray diffraction (XRD). As shown in Figure 4a, the coating on the surface of Ti-ZnP1 and Ti-ZnP2 samples contains both zinc oxide and zinc phosphate crystals.
对样品表面进行X射线光电子能谱(XPS)宽场扫描，得到图4b所示的XPS全谱谱图。其中特征峰的强度代表了表面该元素含量的高低。对比不同组样品的图谱可知，Ti-ZnP1和Ti-ZnP2样品表面增加了磷元素的特征峰。图4c为由XPS结果分析得到的材料表面各个元素的原子百分比，可见Ti和Ti-ZnO样品表面基本不含磷元素，而Ti-ZnP1和Ti-ZnP2样品表面的磷元素含量大大增加，说明在磷酸氢盐中的水热处理能够将氧化锌成功转化成磷酸锌。The sample surface was scanned by X-ray photoelectron spectroscopy (XPS) wide field, and the XPS full spectrum shown in Figure 4b was obtained. The intensity of the characteristic peak represents the content of the element on the surface. Comparing the spectra of different groups of samples, it can be seen that the characteristic peaks of phosphorus are added on the surface of Ti-ZnP1 and Ti-ZnP2 samples. Figure 4c is the atomic percentage of each element on the surface of the material obtained by XPS analysis. It can be seen that the surface of Ti and Ti-ZnO samples basically does not contain phosphorus, while the phosphorus content on the surface of Ti-ZnP1 and Ti-ZnP2 samples is greatly increased, indicating that the hydrothermal treatment in hydrogen phosphate can successfully convert zinc oxide into zinc phosphate.
采用静态水接触角测试仪测试材料表面润湿性。图4d是各组样品的静态水接触角结果。横坐标为样品名称，纵坐标为接触角的度数。由图4d可知，未经处理的Ti样品的接触角为60°左右；在Ti表面制备了氧化锌纳米棒涂层后，接触角降低至22°左右；将氧化锌部分转换成磷酸锌后，接触角进一步降低。The surface wettability of the material was tested using a static water contact angle tester. Figure 4d shows the static water contact angle results of each group of samples. The horizontal axis is the sample name, and the vertical axis is the degree of the contact angle. As shown in Figure 4d, the contact angle of the untreated Ti sample is about 60°; after the zinc oxide nanorod coating is prepared on the Ti surface, the contact angle is reduced to about 22°; after the zinc oxide is partially converted into zinc phosphate, the contact angle is further reduced.
实施例5Example 5
采用划痕实验检测所形成的涂层与基底之间的结合力。在样品表面形成划痕后，使用SEM观察划痕两侧涂层的脱落情况。由图5可见，三种样品划痕边缘都未出现崩裂和大面积脱落的情况，说明本发明所制备的涂层与基底之间具有良好的结合力。The scratch test was used to detect the bonding strength between the formed coating and the substrate. After a scratch was formed on the sample surface, the coating shedding on both sides of the scratch was observed using SEM. As shown in Figure 5, there was no cracking or large-scale shedding at the scratch edges of the three samples, indicating that the coating prepared by the present invention had good bonding strength with the substrate.
实施例6Example 6
将各组样品浸没入Hank’s缓冲液中，保存于37℃，检测样品在模拟体内的生理条件下的锌离子的释放情况。由图6可知，各组样品在30天的检测周期内都可以持续释放出锌离子。其中Ti-ZnO样品的释放速度最快，Ti-ZnP1样品次之，Ti-ZnP2样品释放锌离子的速度最慢。说明将氧化锌部分转换成磷酸锌后，能够显著降低锌离子的释放速率。Each group of samples was immersed in Hank’s buffer and stored at 37°C to detect the release of zinc ions under physiological conditions simulating the body. As shown in Figure 6, each group of samples can continuously release zinc ions within the 30-day test period. Among them, the Ti-ZnO sample has the fastest release rate, followed by the Ti-ZnP1 sample, and the Ti-ZnP2 sample has the slowest release rate of zinc ions. This shows that converting part of zinc oxide into zinc phosphate can significantly reduce the release rate of zinc ions.
实施例7Example 7
将实施例6中所述保存于Hank’s缓冲液中不同时间的样品取出，使用SEM观察样品的表面形貌。如图7所示，Ti-ZnO样品在溶液中浸泡5天后，表面的纳米棒状结构就开始崩塌；浸泡20天后，棒状结构完全消失，并形成孔洞状的腐蚀坑。而Ti-ZnP1和Ti-ZnP2样品在浸泡的30天过程中，表面形貌只有轻微改变。以上结果与实施例6中的结果吻合，说明将氧化锌部分转换成磷酸锌后，能够很大程度上减缓表面涂层的降解速率。The samples stored in Hank’s buffer for different periods of time as described in Example 6 were taken out, and the surface morphology of the samples was observed using SEM. As shown in Figure 7, after the Ti-ZnO sample was immersed in the solution for 5 days, the nanorod-like structure on the surface began to collapse; after immersion for 20 days, the rod-like structure completely disappeared, and a hole-like corrosion pit was formed. However, the surface morphology of the Ti-ZnP1 and Ti-ZnP2 samples changed only slightly during the 30-day immersion process. The above results are consistent with the results in Example 6, indicating that the degradation rate of the surface coating can be greatly slowed down after the zinc oxide is partially converted into zinc phosphate.
实施例8Example 8
将人来源的骨髓间充质干细胞接种并培养于各组样品表面。图8为细胞在样品表面培养1天、3天、5天后使用CCK-8检测试剂盒获得的细胞的增殖情况。其中横坐标为细胞培养天数，纵坐标为检测试剂盒对应孔在450nm波长下的吸光度。吸光度越高表明增殖越快。由结果可见，培养在Ti表面的细胞能够随培养时间的增加正常增殖，而Ti-ZnO表面由于氧化锌较强的细胞毒性，其表面的细胞在培养1天以后即完全丧失活性。Ti-ZnP1样品虽降低了Ti-ZnO样品的毒性但与Ti相比仍一定程度上抑制了细胞活性。而Ti-ZnP2样品表面的细胞与Ti表面的细胞基本相同。以上结果说明随着氧化锌纳米棒涂层中的部分氧化锌转换成了降解速度更慢的磷酸锌，大大降低了材料的生物毒性，并且随着反应时间的延长，磷酸锌的转换比例增加，涂层的细胞毒性也更低。Human bone marrow mesenchymal stem cells were inoculated and cultured on the surface of each group of samples. Figure 8 shows the proliferation of cells obtained using the CCK-8 detection kit after the cells were cultured on the sample surface for 1 day, 3 days, and 5 days. The horizontal axis is the number of days of cell culture, and the vertical axis is the absorbance of the corresponding hole of the detection kit at a wavelength of 450nm. The higher the absorbance, the faster the proliferation. It can be seen from the results that the cells cultured on the Ti surface can proliferate normally with the increase of culture time, while the cells on the Ti-ZnO surface completely lose their activity after 1 day of culture due to the strong cytotoxicity of zinc oxide. Although the Ti-ZnP1 sample reduces the toxicity of the Ti-ZnO sample, it still inhibits cell activity to a certain extent compared with Ti. The cells on the surface of the Ti-ZnP2 sample are basically the same as those on the Ti surface. The above results show that as part of the zinc oxide in the zinc oxide nanorod coating is converted into zinc phosphate, which degrades more slowly, the biological toxicity of the material is greatly reduced, and as the reaction time increases, the conversion ratio of zinc phosphate increases, and the cytotoxicity of the coating is also lower.
实施例9Example 9
使用骨诱导培养基培养各组样品表面的骨髓间充质干细胞3天、7天和14天，使其向成骨细胞分化，然后检测成骨细胞形成早期的标志性酶碱性磷酸酶(ALP)的活性(图9a)、细胞分泌的胶原的情况(图9b)，以及细胞外基质的矿化(图9c)情况。结果中横坐标为细胞诱导天数，图9a纵坐标为ALP的活性，并且其活性使用细胞内总蛋白含量做归一化处理；图9b-c纵坐标为检测试剂盒对应孔在570nm波长下的吸光度，吸光度越高表明细胞外基质矿化程度越高。由结果可知Ti-ZnO样品由于细胞毒性较强，成骨细胞难以在其表面正常生长，更难以向成骨细胞分化；Ti-ZnP1表面培养的细胞具有一定的成骨细胞分化趋势；Ti-ZnP2样品表面培养的细胞ALP活性最高，在诱导21天后胶原分泌最多且细胞外基质的矿化程度最高。以上结果证明Ti-ZnP2能够促进骨髓间充质干细胞向成骨分化。Bone marrow mesenchymal stem cells on the surface of each group of samples were cultured with osteoinduction medium for 3 days, 7 days and 14 days to differentiate into osteoblasts, and then the activity of alkaline phosphatase (ALP), a marker enzyme in the early stage of osteoblast formation (Figure 9a), the collagen secreted by cells (Figure 9b), and the mineralization of extracellular matrix (Figure 9c) were detected. In the results, the horizontal axis is the number of days of cell induction, the vertical axis of Figure 9a is the activity of ALP, and its activity is normalized using the total protein content in the cells; the vertical axis of Figure 9b-c is the absorbance of the corresponding well of the detection kit at a wavelength of 570nm, and the higher the absorbance, the higher the degree of extracellular matrix mineralization. The results show that due to the strong cytotoxicity of Ti-ZnO samples, osteoblasts are difficult to grow normally on their surface, and it is even more difficult to differentiate into osteoblasts; cells cultured on the surface of Ti-ZnP1 have a certain trend of osteoblast differentiation; cells cultured on the surface of Ti-ZnP2 samples have the highest ALP activity, and the most collagen secretion and the highest degree of extracellular matrix mineralization after 21 days of induction. The above results prove that Ti-ZnP2 can promote the osteogenic differentiation of bone marrow mesenchymal stem cells.
实施例10Example 10
在各组样品表面培养细菌培养金黄色葡萄球菌和大肠杆菌来检测样品表面的抗菌性能。由图10a可见，Ti表面无抗菌效果，ZnO表面抗菌效果最好，几乎可以杀灭表面的所有细菌。Ti-ZnP1和Ti-ZnP2表面的抗菌效果虽稍差，但是经计算抗菌率也在90％以上。以上结果证明Ti-ZnP1和Ti-ZnP2样品具有优异的抗感染能力。Staphylococcus aureus and Escherichia coli were cultured on the surface of each group of samples to detect the antibacterial properties of the sample surface. As shown in Figure 10a, the Ti surface has no antibacterial effect, and the ZnO surface has the best antibacterial effect, which can kill almost all bacteria on the surface. Although the antibacterial effect of Ti-ZnP1 and Ti-ZnP2 surfaces is slightly worse, the calculated antibacterial rate is also above 90%. The above results prove that Ti-ZnP1 and Ti-ZnP2 samples have excellent anti-infection ability.
本领域的普通技术人员可以理解，上述各实施方式是实现本申请的具体实施例，而在实际应用中，可以在形式上和细节上对其作各种改变，而不偏离本申请的精神和范围。任何本领域技术人员，在不脱离本申请的精神和范围内，均可作各自更动与修改，因此本申请的保护范围应当以权利要求限定的范围为准。Those skilled in the art can understand that the above-mentioned embodiments are specific examples for implementing the present application, and in practical applications, various changes can be made to them in form and details without departing from the spirit and scope of the present application. Any person skilled in the art can make their own changes and modifications without departing from the spirit and scope of the present application, so the scope of protection of the present application shall be based on the scope defined in the claims.
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| CN202211191672.6ACN115501392B (en) | 2022-09-28 | 2022-09-28 | A zinc oxide/zinc phosphate nanorod composite antibacterial coating and its preparation method and application |
| PCT/CN2022/137052WO2024066040A1 (en) | 2022-09-28 | 2022-12-06 | Zinc oxide/zinc phosphate nanorod composite antibacterial coating and preparation method therefor and use thereof |
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