新型温敏性重组人釉原蛋白载体与传统丙二醇藻酸酯载体的体外测试效外测试效果比较

Comparison of a new thermosensitive rhAm carrier versus traditional PGA carrier for in vitro antibacterial activity and biocompatibility

目的评价新型温敏性重组人釉原蛋白(rhAm)载体和传统丙二醇藻酸酯(PGA)载体的体外表征和对人牙周膜成纤维细胞活性的影响。方法分别利用3.3%PGA与rhAm混合制备PGA-rhAm,2%的壳聚糖(CS)和rhAm混合并使用质量分数60%的β-甘油磷酸钠溶液(βGP)作为交联剂制备CS-βGP-rhAm凝胶;表征通过检测黏度、凝固时间、pH值、溶胀率、体外生物降解率和缓释性能来评估;通过观察金黄色葡萄球菌生长状况比较材料的抗菌效果;生物相容性评估是在人牙周膜成纤维细胞(hPDLFs)上利用CCK8检测各组细胞增殖能力,划痕实验检测细胞迁移能力,RT-qPCR检测成骨基因mRNA表达,Westernblot检测蛋白的表达水平,碱性磷酸酶(ALP)染色检测各组细胞成骨分化情况。结果PGA-rhAm具有黏度值3.262±0.055 Pa.s,CS-βGP-rhAm在37℃下具有6 min凝固成型能力、接近口腔环境的pH值、约90%的溶胀率,同时缓释rhAm可维持2周以上,自身降解时间维持3周以上。CS-βGP负载rhAm相比PGA负载rhAm更有效抑制金黄色葡萄球菌生长(P<0.001)。细胞水平上,CS-βGP是否负载rhAm都可促进细胞增殖(P<0.001),PGA促进细胞增殖效果不显著。划痕实验显示,CS-βGP和PGA负载rhAm后均可促进细胞迁移(P<0.01)。RT-qPCR和Westernblot结果显示,CS-βGP负载rhAm能促进RUNX2、OCN mRNA水平(P<0.001),上调Ki67(P<0.001)、RUNX2(P<0.001)、CollagenⅠ(P<0.01)、β-catenin(P<0.05)蛋白表达。PGA负载rhAm促进RUNX2(P<0.05)、OCN(P<0.01)mRNA水平表达,蛋白水平变化无统计学意义。CS-βGP组ALP染色蓝紫色颗粒数增加,PGA组无明显变化。结论CS-βGP具有能缓释rhAm的性能,同时相比传统的PGA载体,CS-βGP具有温度成型的特点、抑制金黄色葡萄球菌生长的性能、显著提高hPDLFs的生物活性并且负载rhAm后不影响其生物活性。

Objective To compare a new thermosensitive recombinant human amelogenin(rhAm)carrier and traditional propylene glycol alginate(PGA)carrier for their characteristics,antibacterial activity,and biocompatibility with human periodontal membrane fibroblasts.Methods PGA-rhAm was prepared by mixing 3.3%PGA and rhAm,and CS-βGP-rhAm was prepared by mixing 2%chitosan(CS)with rhAm and then with 60%β-sodium glycerophosphate solution(βGP)as the crosslinking agent.The biophysical properties of the prepared carriers were characterized,and their antibacterial activity was assessed by observing Staphylococcus aureus growth.The biocompatibility of the carriers was evaluated in human periodontal membrane fibroblasts(hPDLFs)using CCK8 assay and scratch test,and mRNA and protein expressions of osteogenic genes of the cells incubated with the carriers were detected using RT-qPCR and Western blotting;osteogenic differentiation of the cells was detected using alkaline phosphatase staining.Results PGA-rhAm had a viscosity value of 3.262±0.055 Pa.s.CS-βGP-rhAm had a solidification capacity of 6 min at 37℃with a pH value close to that of the oral cavity and a swelling rate of about 90%.CS-βGP-rhAm maintained sustained release of rhAm for over 2 weeks with a self-degradation time over 3 weeks.CS-βGP-rhAm more effectively inhibited the growth of S.aureus than rhAm-loaded PGA.While PGA did not obviously affect the proliferation of hPDLFs,both CS-βGP and CS-βGP-rhAm significantly promoted the cell proliferation(P<0.001).Scratch test showed that after rhAm loading,both CS-βGP and PGA promoted cell migration(P<0.01).CS-βGP-rhAm significantly enhanced the mRNA expressions of RUNX2 and OCN mRNA level and the protein expressions of Ki67,RUNX2,collagen I,andβ-catenin(P<0.05);PGA-rhAm only enhanced RUNX2(P<0.05)and OCN(P<0.01)mRNA expressions without significant effects on the protein expressions.Alkaline phosphatase staining results showed that CS-βGP,but not PGA,promoted osteogenic differentiation of hPDLFs.Conclusion CS-βGP carrier is capable of sustained release of rhAm,inhibiting the growth of S.aureus,and improving the biological activity of hPDLFs without affecting the bioactivity of rhAm after drug loading.