Preparation and cytocompatibility of cuttlebone-transformed hydroxyapatite

doi:10.3969/j.issn.2095-4344.2014.16.012

Abstract

Abstract:

BACKGROUND: Recently, bone tissue engineering is highlighted from the nature source.

OBJECTIVE: To explore the preparation of a cuttlebone-transformed hydroxyapatite scaffold as the novel bone substitution.

METHODS: As raw materials, the cuttlebone appeared to have a hydrothermal reaction with diammonium phosphate under specific conditions, and the hydrothermal products were characterized respectively by X-ray diffraction, Fourier transform infrared spectroscopy, energy-dispersive X-ray spectroscopy and scanning electron microscopy.

RESULTS AND CONCLUSION: The results showed that the hydrothermal products were hydroxyapatite. The cuttlebone-transformed hydroxyapatite possessed good three-dimensional structure. Cytocompatibility of the cuttlebone-transformed hydroxyapatite with MG63 cells cultured in vitro were observed and we found the cuttlebone-transformed hydroxyapatite had no obvious effects on cell growth and no toxicity, which can be used as new bone tissue engineering scaffolds.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

全文链接：

Key words: hydroxyapatites, durapatite, biocompatible materials, stents

CLC Number:

R318

Huang Xie-shan, Liu Ming, Wang Jiang. Preparation and cytocompatibility of cuttlebone-transformed hydroxyapatite[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(16): 2532-2537.

Figures/Tables 4

2.1 墨鱼骨转化羟基磷灰石材料的制备结果图1A是水热反应前(原墨鱼骨)及不同水热反应时间样品粉末的X射线衍射图谱，图1A中图谱a经X射线衍射图谱处理软件JADE进行分析，得到其各衍射峰的d值与JCPDSNo.05-0453完全一致，表明构成墨鱼骨的矿物是文石相碳酸钙结构。图1中图谱b，c，d，e，f对应样品的水热反应时间分别为2，4，6，8，10 h。用MDI Jade 7.0软件分别对各图谱进行分析，得出在水热反应2 h后，出现了羟基磷灰石物相，其晶型结构也发生了变化。随着反应的进行，墨鱼骨文石矿物相逐步向羟基磷灰石物相转化，图中b，c，d是文石和羟基磷灰石的衍射峰共存。在反应时间4 h时，羟基磷灰石衍射峰强度远高于文石，说明水热反应4 h后，大部分文石相碳酸钙已转化为羟基磷灰石。图中e其各衍射峰与JCPDSNo.74-0566相符，说明在反应时间达到8 h时，文石相碳酸钙已完全转化成羟基磷灰石，图中f图谱与e图谱完全一样，说明水热反应8 h后样品的晶型结构稳定无变化。图1B是水热反应前(原墨鱼骨)及不同水热反应时间样品粉末的傅里叶变换红外光谱图谱。图1B中图谱a与文石相碳酸钙的傅里叶变换红外光谱图相符[34]，表明构成墨鱼骨的矿物是文石相碳酸钙。其CO32-不对称伸缩振动峰(1 450 cm-1强峰)、面外变形振动峰(880 cm-1中强峰)和面内变形振动峰(712 cm-1弱峰)的吸收强度随着水热反应的进行逐渐变弱(图谱b-d)，在反应达到8 h(图谱e)时消失。在反应达到2 h(图谱b)时，出现了P-O振动峰(1 084 cm-1和575 cm-1)和OH-伸缩振动峰(615 cm-1)，其吸收强度随着水热反应时间的进行石的傅里叶红外光谱图谱相符。表明墨鱼骨文石相碳酸钙在磷酸盐溶液水热反应作用下，逐步转化为羟基磷灰石，并且转化程度随着反应时间的延长而增加。文石矿物在磷酸盐溶液中水热转变为羟基磷灰石，通常被认为是发生了局部规整离子交换反应，因为羟基磷灰石(六方晶系，a=0.943 nm，b=0.688 nm，g=1 200)和文石相碳酸钙(a=0.939 nm，b=0.574 nm，g=1 170，可以描述为假六方晶系)的晶格参数相近[34]，离子所处的位置大致相同[35]，当文石相碳酸钙经局部规整离子交换反应转化为羟基磷灰石时，由于减少了成核作用所需结构重排的能量，从而使反应易进行，表部文石相碳酸钙直接与磷酸盐发生水热反应，内部文石相碳酸钙不与溶液直接接触，易与扩散进去的PO43-，OH-离子发生固态局部规整离子交换反应转化成羟基磷灰石，反应从外到内，所以是一个随时间逐步转化的过程。 2.2 墨鱼骨转化羟基磷灰石材料的成分和形貌结构图2A是利用X射线光电子能谱法分析的预处理过的墨鱼骨的X射线能谱法谱图，图2B是利用X射线能谱法分析水热反应 8 h的产物的X射线能谱法谱图，结果显示墨鱼骨的主要化学成分文石相碳酸钙在水热反应8 h的产物中的C元素已经检测不到，检测到元素主要为Ca，O，P元素[36]。X射线能谱法微区定量分析结果如表1所示，通过计算得到水热反应产物的Ca/P为1.67，与羟基磷灰石Ca10(PO4)6(OH)2的化学计量比相符合。说明在水热反应过程中，经磷酸盐溶液作用，反应产物中已不存在文石相碳酸钙，完全转化为羟基磷灰石。"

图3是墨鱼骨转化羟基磷灰石的扫描电镜照片，大体观察为瓷白色，背部呈肉眼可见的多孔状外观，纹理清晰(图3A)。可观察到有较粗大的支柱样结构，支柱之间相互平行，即为肉眼可见之清晰纹理，相邻支柱间是与之相交排列的小梁结构，壁较薄，支柱与小梁相间排列。表面光滑，并呈现出板层样(图3B)，气孔率高达80%以上。构成规则的壁龛样网格(图3C)，由“横墙”和“竖壁”规则搭建而成多孔通道结构，而且这些孔道是相通的，孔道开口大体呈长方形，大小较均匀，尺寸为200-300 μm(图3D)。 2.3 墨鱼骨转化羟基磷灰石的细胞相容性材料与细胞复合培养1 d，光镜观察到材料周边有少量细胞，细胞形态不够清晰，细胞延展的不够充分(图4A)。材料与细胞复合培养3 d，材料周边的细胞数量有所增多，细胞形态趋于清晰，细胞贴材料周边生长，生长状态良好，但生长活力不够旺盛(图4B)。材料与细胞复合培养5 d，细胞数量明显增加，细胞贴附材料生长良好，细胞形态可分辨为梭形、三角形和多边形(图4C)。材料与细胞复合培养7 d，材料周边的细胞数量明显增多，布满材料周边胞浆透明，生长活力旺盛，细胞形态更加清晰，多为梭形、三角形和多边形，无细胞衰老及异常分裂现象(图4D)，可见墨鱼骨转化羟基磷灰石材料不影响细胞正常的增殖。图5是试验组和对照组MG63细胞的MTT检测结果，根据吸光度值随着细胞数的增加而增加，两者呈现明显的正相关关系[37]，各组材料的细胞随着培养时间的延长，数量都有所增加，在各个时间点各组之间细胞的数量差异有显著性意义(P < 0.05)。表2为MG63细胞与材料体外培养1，4和7 d时细胞的相对生长率(细胞相对生长率按下式计算：细胞相对生长率= 其中AE和AC分别是实验组和对照组的吸光度值)。按照GB/T16886.5-2003标准[38]，可以看出材料的细胞毒性为0级，属于无细胞毒性的材料，从细胞相容性方面的研究结果，可知材料对MG63细胞的生长无明显影响且无明显毒性，属于无细胞毒性材料，具有较好的细胞相容性。 "

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