High-performance porous beta-tricalcium phosphate bone tissue engineering scaffolds using 3D printing

doi:10.3969/j.issn.2095-4344.2014.43.005

Abstract

Abstract:

BACKGROUND: Although the preparation of bone tissue engineering scaffolds can achieve satisfactory results by solvent casting/particulate leaching, in situ molding method, electrospinning, phase seperation/freeze drying, gas foaming, there are still some deficiencies in the accuracy, pore uniformity, spatial structure complexity, personalized stents.
OBJECTIVE: To prepare β-tricalcium phosphate bone tissue engineering scaffolds using 3D printing.
METHODS: Drug-loaded β-tricalcium phosphate scaffolds were prepared with 3D printing, and the structure was observed to measure its porosity and mechanical strength. The scaffold was immersed in simulated body fluid for 15 weeks to observe the quality change. The scaffold was co-cultured with rat bone marrow mesenchymal stem cells for 7 days to observe cell adhesion and morphological changes. Rat bone marrow mesenchymal stem cells were cultured in extracts of drug-loaded β-tricalcium phosphate scaffold and low-glucose Dulbecco's modified Eagle’s medium containing 15% fetal bovine serum for 24, 48, and 72 hours, to determine the absorbance values and cytotoxicity grading, respectively. Meanwhile, the cells were subjected to osteogenic culture for 1 week, and
the alkaline phosphatase activities in two groups were detected.
RESULTS AND CONCLUSION: The prepared scaffold showed irregular micropores, high porosity, uniform pore distribution, high pore connectivity rate, and large compressive strength. The drug-loaded β-tricalcium phosphate scaffold degraded completely with 15 weeks, and cancellous bone defect repair was completed in the same period. Rat bone marrow mesenchymal stem cells adhered to the surface of drug-loaded β-tricalcium phosphate scaffold and went deep into the scaffold, showing good growth and proliferation. The activity of alkaline phosphatase was also improved. These findings indicate that the drug-loaded β-tricalcium phosphate scaffold has good biocompatibility.

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: calcium phosphates, tissue engineering, finite element analysis

CLC Number:

R318

Yuan Jing, Zhen Ping, Zhao Hong-bin . High-performance porous beta-tricalcium phosphate bone tissue engineering scaffolds using 3D printing[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(43): 6914-6921.

Figures/Tables 3

2.1 β-磷酸三钙骨组织工程支架的外观结构、表面显微结构及孔隙率制备的β-磷酸三钙立体支架截面结构如图2A所示，正中剖面呈现条束状排列结构，为含有微观孔隙的β-磷酸三钙，宏观孔隙400-500 μm，宏观孔隙相互连通。环境扫描电镜观察显示该材料在支架区有微观多孔隙结构，微孔分布均匀，微观孔隙互相连通，大小相仿，孔径2-8 μm，如图2B所示。经检测，三维多孔β-磷酸三钙支架总孔隙率检测为(61.76±2.53)%，其中宏观孔隙率约为42.5%，微观孔隙率为19.26%。 2.2 烧结后多孔β-磷酸三钙支架材料的物相分析烧结后的β-磷酸三钙支架X射线衍射分析图谱见图3B，纳米β-磷酸三钙粉末用同样方法获得X射线衍射分析图谱见图3A。经过对2个图谱主要波峰对比可知，样品烧结前后物相组成没有明显变化，经过1 100 ℃煅烧后支架的物相组成为β-磷酸三钙，没有出现相转变，证明1 100 ℃高温烧结2 h后得到了结晶度较高的β-磷酸三钙多孔支架材料。 2.3 多孔β-磷酸三钙支架抗压强度及有限元分析取烧结后的β-磷酸三钙支架通过抗压强度测试，最大压缩强度为(3.31±0.64)MPa，力学性能可以达到松质骨的抗压强度2-12 MPa[19]。随机抽取1支架的压缩应力-应变曲线见图4A，曲线上的第一个峰为支架的抗压强度。β-磷酸三钙支架第一主应力分布云图显示，当第一主应力作用时受力最大的部位在材料中部，见图4B。 2.4 模拟体液pH值变化和组织工程支架的质量变化前4周，浸有样品的模拟体液pH值变化不大，pH值在7.4左右；而从第4周到第6周，模拟体液的pH值呈直线下降，pH值在5.5左右，说明支架降解产生的酸性降解产物主要在这个阶段从支架扩散到模拟溶液中；15周时，pH值略有升高，说明材料降解趋于完毕，出现了pH有所回升的现象，变化情况见图5A。在体外降解过程中，材料质量随着时间变化而逐渐降低，在15周时材料降解质量趋于0，降解曲线见图5B。 2.5 活细胞增殖率与成骨活性测定结果实验组与阴性对照组细胞碱性磷酸酶活性分别为(63.4±3.4)，(143.3±1.4)金氏单位/L，组间比较差异有显著性意义(P < 0.05)。MTT 检测结果见表1。实验组细胞24，48，72 h的细胞相对增殖率分别为107%，104%，104%，对应的细胞毒性级别均为0。实验组材料上生长良好，有少量细胞长入孔隙，实验组与阴性对照组相比无差异。 2.6 多孔β-磷酸三钙支架上细胞黏附与生长形态观察显微镜观察材料边缘细胞情况观察：实验组24 h后，见材料边缘有大鼠骨髓间充质干细胞，生长态势良好，细胞较稀疏，三四个细胞簇拥在一起，见图6A；培养48 h后，见材料边缘细胞数量显著增加，布满整个视野，细胞之间紧挨，生长旺盛，并在材料边缘有向上生长，有的细胞进入支架内部，见图6B；72 h后，见细胞有重叠生长，细胞向材料空间内部生长，细胞生长旺盛，在材料边界细胞生长密集，见图6C。通过单位面积计数，发现实验组24，48，72 h细胞数量和阴性对照组无明显差异，两组细胞形态、细胞之间距离均无明显差异。金相显微镜观察支架材料表面细胞：共同培养48 h取出材料后，荧光染料CFSE对支架材料进行染色，见图7，可见细胞生长于多孔材料表面和孔隙中。扫描电镜观察材料表面：共同培养48 h，可见在β-磷酸三钙孔隙有大鼠骨髓间充质干细胞，细胞呈梭形和不规则形，与材料表面黏附，在材料表面细胞贴壁、密集生长，长势活跃，并伸出伪足附着材料表面，见图8。"

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