3D打印n-HA/壳聚糖/米诺环素复合支架的构建与其理化性能、细胞毒性研究

Construction,physicochemical properties and cytotoxicity of 3D printed n-HA/chitosan/minocycline composite scaffold

目的采用3D打印的方法构建不同质量的纳米羟基磷灰石(Nano-hydroxyapatite,n-HA)/壳聚糖/米诺环素的载药复合支架,并进行相关性能测试。方法根据加入不同质量的羟基磷灰石和不同浓度的米诺环素,经组合,实验组分为S1(n-HA 2.5 g、米诺环素1μmol/L)、S2(n-HA 3.5 g、米诺环素1μmol/L)和S3(n-HA 4.5 g、米诺环素1μmol/L);S4(n-HA 2.5 g、米诺环素5μmol/L)、S5(n-HA 3.5 g、米诺环素5μmol/L)和S6(n-HA 4.5 g、米诺环素5μmol/L);S7(n-HA 2.5 g、米诺环素10μmol/L)、S8(n-HA 3.5 g、米诺环素10μmol/L)和S9(n-HA 4.5 g、米诺环素10μmol/L)。对照组分为D1(n-HA 2.5 g、米诺环素0μmol/L)、D2(n-HA 3.5 g、米诺环素0μmol/L)和D3(n-HA 4.5 g、米诺环素0μmol/L)。扫描电镜下观察支架形貌结构,对复合支架的吸水率、力学性能、孔径、pH值、体外降解速率、MTT细胞毒性进行检测。结果不同质量的纳米羟基磷灰石和米诺环素均对复合支架的性能产生影响,支架的孔隙率和孔径会随纳米羟基磷灰石和米诺环素质量的增加不断减小;随着n-HA质量的增加,吸水率呈逐渐下降趋势,但对照组间差异无统计学意义(P=0.896),同一n-HA质量下实验组的吸水率随着米诺环素浓度的增高缓慢增加(P=0.0009),但随着时间的延长,各实验组吸水率又慢慢降低;随着n-HA质量的增加,对照组支架的拉伸强度和最大载荷均有不同程度的降低(P=0.0002)。实验组S1-S6复合支架拉伸强度随米诺环素增加而增大,当米诺环素的浓度持续加至10μmol/L后,S7、S8、S9组拉伸强度反而降低;相同n-HA质量下的实验组pH值随米诺环素浓度的增加在降低(P=0.003),S8组pH值接近健康牙周组织;随着米诺环素浓度的增加,降解率降低,随着n-HA质量的增加,降解率反而增加;不同质量的n-HA支架的细胞增殖率差异无统计学意义,浓度为1、5μmol/L米诺环素实验组与对照组差异无统计学意义(P=0.65),当米诺环素浓度为10μmol/L时,实验组OD值在3 d后出现抑制细胞增殖的情况(P=0.007)。结论实验中的米诺环素质量为1、5μmol/L时,复合支架组具有良好的物理化学性能,无细胞毒性。

Objective The drug-carrying composite scaffolders of Nano-hydroxyapatite(Nano-hydroxyapatite,n-HA)/chitosan/minocycline with different quality were constructed by 3D printing method,and the related performance tests were carried out.MethodsBased on the addition of different masses of hydroxyapatite and different concentrations of minocycline,by the combination,the experimental group was divided into S1(n-HA 2.5 g,minocycline 1μmol/L),S2(n-HA 3.5 g,minocycline 1μmol/L)and S3(n-HA 4.5 g,minocycline 1μmol/L);S4(n-HA 2.5 g,minocycline 5μmol/L),S5(n-HA 3.5 g,minocycline 5μmol/L),and S6(n-HA 4.5 g,minocycline 5μmol/L);S7(n-HA 2.5 g,minocycline 10μmol/L),S8(n-HA 3.5 g,minocycline 10μmol/L),and S9(n-HA 4.5 g,minocycline 10μmol/L).The control group was D1(n-HA 2.5 g,minocycline 0μmol/L),D2(n-HA 3.5 g,minocycline 0μmol/L),and D3(n-HA 4.5 g,minocycline 0μmol/L).The morphological structure of the scaffold was observed under the scanning electron microscope,and the water absorption,mechanical properties,pore size,pH,degradation experiments and MTT cytotoxicity of the composite scaffold was examined.ResultsThen-HA and minocycline with different qualities had an impact on the performance of the composite scaffold.The porosity and pore size of the scaffold decreased with the increase of n-HA and minocycline.With the increase of n-HA quality,water absorption showed a gradual downward trend,but there was no statistical significance between controls(P=0.896).Under the same n-HA quality,water absorption of the experimental groupincreased slowly with the increase of minocycline concentration(P=0.0009),but with the extension of time,water absorption of all experimental groups gradually decreased.With the increase of n-HA mass,the tensile strength and maximum load of the stent in the control group were decreased to varying degrees(P=0.0002).In the experimental group,the tensile strength of S1-S6 composite scaffold increased with the increase of minocycline,but the tensile strength of S7,S8 and S9 groups decreased when minocycline concentration was continuously increased to 10μmol/L.The pH value of experimental group with the same n-HA mass decreased with the increase of minocycline concentration(P=0.003),and the pH value of the S8 group was close to the healthy periodontal tissue.With the increase of minocycline concentration,the degradation rate decreased,and with the increase of n-HA quality,the degradation rate increased.There was no statistically significant difference in the cell proliferation rate of n-HA scaffold with different quality,and there was no statistically significant difference between the minocycline group and the control group when the minocycline concentration was 1 and 5μmol/L(P=0.65).When the minocycline concentration was 10μmol/L,the OD value of the experimental group inhibited cell proliferation after 3 days(P=0.007).ConclusionWhen the concentration of minocycline was 1 and 5μmol/L,the composite scaffold group had good physicochemical properties and no cytotoxicity.