Department of Prosthodontics, School of Dentistry, Faculty of Health Sciences, Aristotle University of Thessaloniki (A.U.Th), Greece.
Department of Materials Science and Technology, University of Crete, Greece.
Dent Mater. 2021 Jan;37(1):e23-e36. doi: 10.1016/j.dental.2020.09.021. Epub 2020 Nov 15.
Hybrid chitosan/gelatin/nanohydroxyapatite (CS/Gel/nHA) scaffolds have attracted considerable interest in tissue engineering (TE) of mineralized tissues. The present study aimed to investigate the potential of CS/Gel/nHA scaffolds loaded with dental pulp stem cells (DPSCs) to induce odontogenic differentiation and in vitro biomineralization.
CS/Gel/nHA scaffolds were synthesized by freeze-drying, seeded with DPSCs, and characterized with flow cytometry. Scanning Electron Microscopy (SEM), live/dead staining, and MTT assays were used to evaluate cell morphology and viability; real-time PCR for odontogenesis-related gene expression analysis; SEM-EDS (Energy Dispersive X-ray spectroscopy), and X-ray Diffraction analysis (XRD) for structural and chemical characterization of the mineralized constructs, respectively.
CS/Gel/nHA scaffolds supported viability and proliferation of DPSCs over 14 days in culture. Gene expression patterns indicated pronounced odontogenic shift of DPSCs, evidenced by upregulation of DSPP, BMP-2, ALP, and the transcription factors RunX2 and Osterix. SEM-EDS showed the production of a nanocrystalline mineralized matrix inside the cell-based and - to a lesser extent - the cell-free constructs, with a time-dependent production of net-like nanocrystals (appr. 25-30nm in diameter). XRD analysis gave the crystallite size (D=50nm) but could not distinguish between the initially incorporated and the biologically produced nHA.
This is the first study validating the potential of CS/Gel/nHA scaffolds to support viability and proliferation of DPSCs, and to provide a biomimetic microenvironment favoring odontogenic differentiation and in vitro biomineralization without the addition of any inductive factors, including dexamethasone and/or growth/morphogenetic factors. These results reveal a promising strategy towards TE of mineralized dental tissues.
壳聚糖/明胶/纳米羟基磷灰石（CS/Gel/nHA）杂化支架在矿化组织的组织工程（TE）中引起了相当大的关注。本研究旨在探讨负载牙髓干细胞（DPSCs）的 CS/Gel/nHA 支架在诱导牙向分化和体外生物矿化方面的潜力。
通过冷冻干燥合成 CS/Gel/nHA 支架，接种 DPSCs，并通过流式细胞术进行表征。扫描电子显微镜（SEM）、活/死染色和 MTT 测定用于评估细胞形态和活力；实时 PCR 用于牙向分化相关基因表达分析；SEM-EDS（能量色散 X 射线能谱仪）和 X 射线衍射分析（XRD）分别用于矿化结构的结构和化学特性分析。
CS/Gel/nHA 支架在培养 14 天内支持 DPSCs 的存活和增殖。基因表达模式表明 DPSCs 表现出明显的牙向分化，表现为 DSPP、BMP-2、ALP 和转录因子 RunX2 和 Osterix 的上调。SEM-EDS 显示在细胞内和在一定程度上在无细胞构建体中产生了纳米晶矿化基质，并且随着时间的推移产生了网状纳米晶体（直径约为 25-30nm）。XRD 分析给出了晶粒尺寸（D=50nm），但无法区分最初掺入的和生物产生的 nHA。
这是第一项验证 CS/Gel/nHA 支架支持 DPSCs 的存活和增殖的潜力，并提供有利于牙向分化和体外生物矿化的仿生微环境的研究，而无需添加任何诱导因子，包括地塞米松和/或生长/形态发生因子。这些结果揭示了一种有前途的矿化牙组织 TE 策略。
