School of Dentistry, The University of Queensland, Oral Health Centre, Herston, Brisbane, Australia; Institute of Health and Biomedical Innovation, Queensland University of Technology, 60 Musk Avenue, 4059 Kelvin Grove, Australia.
Department of Basic Science, Biology Unit, Deanship of Preparatory Year and Supporting studies, Imam Abdulrahman Bin Faisal University (University of Dammam), Saudi Arabia.
Arch Oral Biol. 2018 Apr;88:67-76. doi: 10.1016/j.archoralbio.2018.01.014. Epub 2018 Jan 31.
Decellularization aims to harness the regenerative properties of native extracellular matrix. The objective of this study was to evaluate different methods of decellularization of periodontal ligament cell sheets whilst maintaining their structural and biological integrity.
Human periodontal ligament cell sheets were placed onto melt electrospun polycaprolactone (PCL) membranes that reinforced the cell sheets during the various decellularization protocols. These cell sheet constructs (CSCs) were decellularized under static/perfusion conditions using a) 20 mM ammonium hydroxide (NH4OH)/Triton X-100, 0.5% v/v; and b) sodium dodecyl sulfate (SDS, 0.2% v/v), both +/- DNase besides Freeze-thaw (F/T) cycling method. CSCs were assessed using a collagen quantification assay, immunostaining and scanning electron microscopy. Residual fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) were assessed with Bio-plex assays.
DNA removal without DNase was higher under static conditions. However, after DNase treatment, there were no differences between the different decellularization methods with virtually 100% DNA removal. DNA elimination in F/T was less efficient even after DNase treatment. Collagen content was preserved with all techniques, except with SDS treatment. Structural integrity was preserved after NH4OH/Triton X-100 and F/T treatment, while SDS altered the extracellular matrix structure. Growth factor amounts were reduced after decellularization with all methods, with the greatest reduction (to virtually undetectable amounts) following SDS treatment, while NH4OH/Triton X-100 and DNase treatment resulted in approximately 10% retention.
This study showed that treatment with NH4OH/Triton X-100 and DNase solution was the most efficient method for DNA removal and the preservation of extracellular matrix integrity and growth factors retention.
脱细胞化旨在利用天然细胞外基质的再生特性。本研究的目的是评估不同的牙周膜细胞片脱细胞方法，同时保持其结构和生物完整性。
将人牙周膜细胞片置于熔融静电纺聚己内酯（PCL）膜上，在各种脱细胞化方案中增强细胞片。这些细胞片构建体（CSC）在静态/灌注条件下使用 a）20mM 氨/曲拉通 X-100，0.5%v/v；和 b）十二烷基硫酸钠（SDS，0.2%v/v），均在 +/- 脱氧核糖核酸酶的条件下，以及冻融（F/T）循环法进行脱细胞化。使用胶原蛋白定量测定、免疫染色和扫描电子显微镜评估 CSC。用 Bio-plex 测定法评估残留的成纤维细胞生长因子（bFGF）、血管内皮生长因子（VEGF）和肝细胞生长因子（HGF）。
无脱氧核糖核酸酶的静态条件下 DNA 去除率更高。然而，经脱氧核糖核酸酶处理后，不同脱细胞化方法之间几乎没有差异，DNA 去除率达到 100%。即使经脱氧核糖核酸酶处理，F/T 中的 DNA 消除效率较低。除 SDS 处理外，所有技术均能保留胶原蛋白含量。NH4OH/Triton X-100 和 F/T 处理后保持结构完整性，而 SDS 处理改变了细胞外基质结构。所有方法脱细胞化后生长因子量减少，SDS 处理后减少最多（几乎检测不到），而 NH4OH/Triton X-100 和脱氧核糖核酸酶处理后保留约 10%。
本研究表明，NH4OH/Triton X-100 和脱氧核糖核酸酶溶液处理是去除 DNA 和保持细胞外基质完整性和生长因子保留最有效的方法。
