Low-temperature synthesis and stabilization of carboxymethyl chitosan stabilized amorphous calcium phosphate

doi:10.3969/j.issn.2095-4344.0893

Abstract

Abstract:

BACKGROUND: Amorphous calcium phosphate can serve as a precursor phase of nano-hydroxyapatite and the storage source for calcium and phosphate. However, the stability of amorphous calcium phosphate is greatly influenced by synthetic environments, which is easy to trigger a phase transition.

OBJECTIVE: To stabilize amorphous calcium phosphate by carboxymethyl chitosan (CMCS) and to investigate the lowest threshold of CMCS with stabilizing capacity and the effects of aging time on stabilization period.

METHODS: CMCS-amorphous calcium phosphate precipitates in different CMCS/Ca molar ratios (1:1, 1:2, 1:3, 1:4, 1:5) were synthesized at 4 ℃. Their microstructures were observed by scanning electron microscopy (SEM) and chemical compositions were analyzed by powder X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR). The lowest threshold of CMCS/Ca with stabilizing capacity was thus acquired. The stable complexes at this lowest threshold were investigated by thermogravimetric analysis and differential scanning calorimetry to determine the content of amorphous calcium phosphate. CMCS-amorphous calcium phosphate complexes with the lowest threshold of CMCS/Ca were then synthesized under different aging times (10, 30, 60, 90 minutes) and dissolved in simulated body fluid. Effects of aging time on stabilization period were discussed following the investigation on the phase transition of amorphous calcium phosphate by SEM and XRD.

RESULTS AND CONCLUSION: (1) According to the results from XRD and FTIR, CMCS-amorphous calcium phosphate precipitates were amorphous when CMCS/Ca ≥ 1:3. (2) According to the findings of SEM, plenty of evenly distributed amorphous calcium phosphate particles were found in the precipitates when CMCS/Ca ≥ 1:3. (3) The lowest threshold of CMCS/Ca used for synthesizing the stable CMCS-amorphous calcium phosphate complexes at 4 ℃ reached to 1:3, and the actual content of amorphous calcium phosphate was 63.63%. (4) According to the results from XRD and SEM, the CMCS-amorphous calcium phosphate complexes dissolved in the simulated body fluid were amorphous under 10, 30, 60 minutes of aging and the corresponding stabilization period was 2, 6, 8 hours, respectively, while the amorphous complexes became crystals under 90 minutes of aging. All these findings indicate that the lowest threshold of CMCS/Ca with the stable CMCS-amorphous calcium phosphate in a low temperature is 1:3. When synthesized with this lowest threshold of CMCS/Ca, the stabilization period of amorphous calcium phosphate is positively correlated with aging time and varies from 2 to 8 hours.

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

Key words: Nanotubes, Carbon, Excipients, Calcium Phosphates, Tissue Engineering

CLC Number:

R318

Wang Ping-ting, Qi Xing-ying . Low-temperature synthesis and stabilization of carboxymethyl chitosan stabilized amorphous calcium phosphate[J]. Chinese Journal of Tissue Engineering Research, doi: 10.3969/j.issn.2095-4344.0893.

Figures/Tables 6

2.1 CMCS-无定形磷酸钙的结构与成分分析 6组冻干沉淀物及CMCS对照的X射线衍射图如图1A所示。CMCS对照组(a)可见2θ在20°附近存在CMCS的典型衍射峰，而在冻干沉淀物组中，随着CMCS/Ca比值降低，由1∶1组至1∶5组CMCS典型衍射峰逐渐降低。同时，1∶1组(b)、1∶2组(c)、1∶3组(d)中在30°左右可见非晶体衍射峰，而当CMCS/Ca比值降至1∶4(e)和1∶5(f)时出现了与无CMCS的CaP组(g)相似的晶体衍射峰。 6组冻干沉淀物及CMCS对照的红外光谱图如图1B所示，CMCS的伸缩振动峰在1 400 cm-1 和1 650 cm-1处(a)，而在CMCS-CaP冻干沉淀物中这两个峰值均发生变化；在CMCS/Ca比值≥1∶3时，在550 cm-1(ν4 PO43-弯模)附近可见单一的光环峰(b-d)；而当CMCS/Ca比值<1∶3或无CMCS时，该单一峰分裂为530 cm-1 和570 cm-1附近的两个峰(e-g)。图2显示了6组冻干沉淀物在扫描电镜下的显微结构，无CMCS辅助合成的CaP呈薄片状晶体结构，片状晶体间无泥雪样物质存在(图2F)；而CMCS-CaP冻干沉淀物各组均可见CaP以大量不规则形状的颗粒形式存在，颗粒间存在泥雪样物质(CMCS)；CaP颗粒尺寸不随CMCS/Ca比值改变而发生可见变化，但随着CMCS/Ca比值降低，颗粒间的泥雪样物质逐渐减少至消失(图2A-E)；当CMCS/Ca比值降低至1∶3时，CaP颗粒的轮廓变得模糊，同时可见少量晶体结构出现(图2C)；当CMCS/Ca比值降低至1∶4时这一趋势更为明显(图2D)；当CMCS/Ca比值进一步降低至1∶5时，可见大量晶体结构生成，不规则CaP颗粒及泥雪样物质显著减少(图2E)。对于CMCS进行热重量及差热分析，在60 ℃附近观察到放热峰值，相应有19.17%的质量降低；加热到280 ℃时再次出现放热峰，同时重量也急剧减少(图3A)；当温度升至1 000 ℃时剩余量降至为7.04%。显示了CMCS/Ca比为1∶3的CMCS-无定形磷酸钙复合物的热重量分析及差热分析结果，放热初期即有16.22%的质量丢失，放热峰值也降低到260 ℃附近；当加热到1000 ℃时CMCS-无定形磷酸钙复合物剩余量为53.31%(图3B)。根据以上提及的数据计算可得CMCS-无定形磷酸钙复合物中无定形磷酸钙的实际量为63.63%。"

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