基于快速成形的磷酸钙多孔陶瓷成骨性能初步研究

OSTEOGENIC ACTIVITY OF POROUS CALCIUM PHOSPHATE CERAMICS FABRICATED BY RAPID PROTOTYPING

目的磷酸钙生物材料由于其优良的生物相容性而具有广阔的应用前景,但传统方法难以制备具有复杂形状的支架。以快速成形方法制备磷酸钙多孔陶瓷支架材料,并对其体外成骨性能进行初步研究。方法采用快速成形方法制备磷酸钙多孔陶瓷支架材料(直径10mm、高度5mm、孔径800μm),取Beagle犬髂骨髓分离培养BMSCs,接种第3代BMSCs于支架材料上。将实验分为4组,A组为成骨诱导培养的细胞/材料复合物组,B组为未经成骨诱导培养的细胞/材料复合物组,另设平皿成骨诱导培养及常规培养为阳性对照组(C组)及阴性对照组(D组)。于接种后第1、3、7天,利用DNA定量检测方法及ALP定量、定性检测方法检测BMSCs在支架材料上的增殖及ALP的表达;并经DiO荧光染色后,应用激光共聚焦显微镜观察BMSCs在材料上的黏附生长情况。结果 DNA定量结果表明,随培养时间增加,C、D组在第3天时细胞生长进入平台期;A、B组细胞呈持续增长趋势,细胞培养过程中增殖速度均略低于C、D组,且未出现明显的平台期。DiO荧光染色示,BMSCs在以快速成形方法制备的磷酸钙多孔陶瓷支架材料上黏附、生长状态良好。ALP染色示,随培养时间延长,A、B组支架上细胞染色均逐渐加深,A组各时间点染色程度均明显强于B组。培养后第1、3天,4组ALP表达均较低,差异无统计学意义(P>0.05);培养第7天,各组ALP表达均明显升高,A组比其他各组明显高表达(P<0.05),B组及C组均明显高于D组(P<0.05)。结论快速成形的多孔磷酸钙陶瓷具有良好的细胞相容性,BMSCs与支架复合在体外诱导培养可以进行成骨分化。因此通过快速成形技术制备的磷酸钙多孔陶瓷是骨组织工程可选的细胞支架。

Objective Calcium phosphate bioceramics has a broad application prospect because of good biocompatibility,but porous scaffolds with complex shape can not be prepared by the traditional methods.To fabricate porous calcium phosphate ceramics by rapid prototyping and to investigate the in vitro osteogenic activities.Methods The porous calcium phosphate ceramics was fabricated by rapid prototyping.The bone marrow mesenchymal stem cells（BMSCs） were isolated from bone marrow of Beagle canine,and the 3rd passage BMSCs were seeded onto the porous ceramics.The cell/ceramics composite cultured in osteogenic medium were taken as the experimental group（group A） and the cell/ceramics composite cultured in growth medium were taken as the control group（group B）.Meanwhile,the cells seeded on the culture plate were cultured in osteogenic medium or growth medium respectively as positive control（group C） or negative control（group D）.After 1,3,and 7 days of culture,the cell proliferation and osteogenic differentiation on the porous ceramics were evaluated by DNA quantitative analysis,histochemical staining and alkaline phosphatase（ALP） activity.After DiO fluorescent dye,the cell adhesion,growth,and proliferation on the porous ceramics were also observed by confocal laser scanning microscope（CLSM）.Results DNA quantitative analysis results showed that the number of BMSCs in all groups increased continuously with time.Plateau phase was not obvious in groups A and B,but it was clearly observed in groups C and D.The CLSM observation indicated that the activity of BMSCs was good and the cells spread extensively,showing good adhesion and proliferation on the porous calcium phosphate ceramics prepared by rapid prototyping.ALP quantitative analysis results showed that the stain of cells on the ceramics became deeper and deeper with time in groups A and B,the staining degree in group A were stronger than that in group B.There was no significant difference in the change of the ALP activity among 4 groups at the first 3 days（P 0.05）;the ALP activity increased obviously in 4 groups at 7 days,group A was significantly higher than other groups（P 0.05） and groups C,D were significantly higher than group D（P 0.05）.Conclusion The porous calcium phosphate ceramics has good cytocompatibility and the designed pores are favorable for cell ingrowth.The porous ceramics fabricated by rapid prototyping has prominent osteogenic differentiation activity and can be used as a choice of scaffolds for bone tissue engineering.