负载羟基磷灰石的PLGA支架材料在骨缺损修复中的研究

On bone defect repair with PLGA-HAP composite scaffolds biomaterials

摘要:

目的：通过检测大鼠骨髓干细胞（bone mesenchymal stem cells，BMSCs）在负载羟基磷灰石的聚乳酸-羟基乙酸共聚物（polylactic-co-glycolic acid，PLGA）支架上的生物学行为，为进一步制备骨修复材料提供实验依据。方法：体外仿生矿化形成不同浓度羟基磷灰石（hydroxyapatite，HAP）PLGA支架材料PLGA-HAP（0 mmol/L、4.2 mmol/L、25 mmol/L）。大鼠BMSCs分别接种其中，检测各组材料对细胞黏附、增殖及成骨分化的影响。结果：在细胞增殖实验中，对照组在1 d、4 d、7 d的光密度值分别为0.361±0.009、0.408±0.005和0.430±0.006，PLGA-HAP（0 mmol/L）支架在1 d、4 d、7 d的光密度值分别为0.203±0.003、0.477±0.006和0.606±0.007，PLGA-HAP（4.2 mmol/L）复合支架在1 d、4 d、7 d的光密度值分别为0.262±0.006、0.527±0.005和0.635±0.003，PLGA-HAP（25 mmol/L）复合支架在1 d、4 d、7 d的光密度值分别为0.270±0.002、0.575±0.004和0.667±0.005，时间因素的F值为10 632.000，HAP浓度因素的F值为678.000，HAP浓度与时间因素交互的F值为732.600，P值均为0.000，数据有统计学差异，说明时间相对较长、矿化程度较高的支架更利于细胞的黏附、生长增殖，时间因素的影响相对积极；在碱性磷酸酶活性值测试中，3 d时复合支架PLGA-HAP（0 mmol/L）、PLGA-HAP（4.2 mmol/L）、PLGA-HAP（25 mmol/L）的活性值分别为0.088±0.015、0.122±0.016和0.125±0.008，7 d时复合支架PLGA-HAP（0 mmol/L）、PLGA-HAP（4.2 mmol/L）、PLGA-HAP（25 mmol/L）的活性值分别为0.146±0.004、0.469±0.024和0.573±0.013，时间因素的F值为1 653.000，HAP浓度因素的F值为400.900，HAP浓度与时间因素交互的F值为280.500，P值均为0.000，数据有统计学差异，说明在相对的一段时间内，不同矿化程度的支架材料均能促进骨缺损修复，随着时间的推移，HAP浓度即矿化程度越高的支架越利于细胞的成骨分化，其中，时间因素的影响相对积极。结论：负载羟基磷灰石的PLGA复合支架材料能够显著促进细胞黏附、增殖及成骨分化的能力，本研究是进一步研制相关生物活性骨修复材料的重要实验依据和基础。

Abstract:

Objective：To provide experimental basis for the further preparation of bioactive bone repair materials by detecting the biological behaviour of rat bone mesenchymal stem cells（BMSCs） in the PLGA-HAP composite scaffolds biomaterials. Methods：Hydroxyapatite（HAP） with different concentrations formed by biomimetic mineralization in vitro and deposited on polylactic-co-glycolic acid（PLGA） turned to PLGA-HAP scaffolds biomaterials（0 mmol/L，4.2 mmol/L，25 mmol/L）. Rat BMSCs were co-cultured into them to detect the effects of each group of materials on cell adhesion，proliferation and osteogenic differentiation. Results：In the cell proliferation experiment，the absorbance values of the control group on 1 d，4 d，and 7 d were 0.361±0.009，0.408±0.005，and 0.430±0.006，the values of PLGA-HAP（0 mmol/L） composite scaffolds on 1 d，4 d，and 7 d were 0.203±0.003，0.477±0.006，and 0.606±0.007，the values of PLGA-HAP（4.2 mmol/L） composite scaffolds at 1 d，4 d，and 7 d were 0.262±0.006，0.527±0.005，and 0.635±0.003，respectively，and PLGA-HAP（25 mmol/L） composite scaffolds at 1 d，4 d，and 7 d values were 0.270±0.002，0.575±0.004，and 0.667±0.005，respectively. The F value of the time factor was 10 632.000，that of the HAP concentration factor was 678.000，and that of the interaction factor was 732.600，all P=0.000. The data had statistical differences，indicating that the scaffold with relatively long time and high mineralization degree was more conducive to cell adhesion，growth and proliferation，and the influence of time factor was relatively positive. In the alkaline phosphatase（ALP） activity value test，the activity values of the composite scaffolds PLGA-HAP（0 mmol/L），PLGA-HAP（4.2 mmol/L），and PLGA-HAP（25 mmol/L） at 3 days were 0.088±0.015，0.122±0.016 and 0.125±0.008，the activity values at 7 days were 0.146±0.004，0.469±0.024 and 0.573±0.013，respec－tively. The F value of the time factor was 1 653.000，the F value of the HAP concentration factor was 400.900，and the F value of the interaction factor was 280.500，all P=0.000. The statistically different data showed that scaffold materials with different mineralization degrees could promote the repair of bone defects in a relative period of time. With the passage of time，the scaffold with higher HAP concentration，i.e. higher mineralization degree，was more conducive to the osteogenic differentiation of cells，and the influence of time was relatively positive. Conclusion：Hydroxyapatite-loaded PLGA composite scaffold can significantly promote cell adhesion，prolifer－ation and osteogenic differentiation. This study is an important experimental basis and foundation for further development of related bioactive bone repair materials.