Objective

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.

Methods

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.

Results

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. 25−30 nm in diameter). XRD analysis gave the crystallite size (D = 50 nm) but could not distinguish between the initially incorporated and the biologically produced nHA.

Significance

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.

中文翻译：

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壳聚糖/明胶/纳米羟基磷灰石混合支架促进牙髓干细胞的牙源性分化和体外生物矿化

目的

混合壳聚糖/明胶/纳米羟基磷灰石（CS / Gel / nHA）支架在矿化组织的组织工程（TE）中引起了相当大的兴趣。本研究旨在研究装载牙髓干细胞（DPSC）的CS / Gel / nHA支架诱导牙源性分化和体外生物矿化的潜力。

方法

通过冷冻干燥合成CS / Gel / nHA支架，用DPSC接种，并用流式细胞仪进行表征。扫描电子显微镜（SEM），活/死染色和MTT分析用于评估细胞形态和生存力。实时PCR用于牙源性相关基因表达分析；SEM-EDS（能量分散X射线光谱法）和X射线衍射分析（XRD）分别用于对矿化构造物的结构和化学特征进行表征。

结果

CS / Gel / nHA支架可支持培养14天以上的DPSC的活力和增殖。基因表达模式表明DPSC的牙源性转移明显，由DSPP，BMP-2，ALP以及转录因子RunX2和Osterix的上调证明。SEM-EDS显示出在基于细胞的细胞内以及（在较小程度上）无细胞结构内产生了纳米晶矿化基质，并随时间产生了网状纳米晶体（直径约25-30 nm）。 。XRD分析给出了微晶尺寸（D  ＝ 50nm），但是不能区分最初掺入的和生物产生的nHA。

意义

这是第一项验证CS / Gel / nHA支架支持DPSC活力和增殖，并提供有利于牙源性分化和体外生物矿化的仿生微环境而无需添加任何诱导性因素（包括地塞米松和/或生长）的潜力的第一项研究/形态发生因素。这些结果揭示了一种理想的矿化牙齿组织TE策略。