Gelatin fibrous scaffolds promote fibrogenic differentiation of human dental pulp stem cells

doi:10.3969/j.issn.2095-4344.0495

Abstract

Abstract:

BACKGROUND: A tooth can be led to lose viability, split easily and miss immune defensive response by pulpitis and pulp necrosis. Determining how to achieve dental pulp regeneration has become a research focus in dentistry. The physicochemical properties and biocompatibility of scaffold materials are crucial for proliferation and differentiation of stem cells.
OBJECTIVE: To study whether a gelatin scaffold can induce dental pulp stem cells to differentiate into fibroblasts.
METHODS: Gelatin scaffolds at different concentrations were prepared by electrospinning method. The surface morphology and physical properties of gelatin scaffolds were tested by using scanning electron microscope and tensile tests. The human dental pulp stem cells (hDPSCs) were seeded on the scaffolds and the cell proliferation and fibrogenic differentiation were tested using MTT and RT-PCR.
RESULTS AND CONCLUSION: The fiber diameter of the 7.5% gelatin scaffold was (2.02±0.36) μm, and it was increased to (3.15±0.52) μm after cross-linking. In the 15% gelatin scaffold, fiber bonding was detected and strengthened until the emergence of flat structures after cross-linking. Both 7.5% and 15% gelatin scaffolds could promote the adhesion and growth of hDPSCs. On day 7, the cell number on the 7.5% gelatin scaffold was significantly higher than that on the 15% gelatin scaffold (P < 0.05). The levels of Collagen I, α-SMA, Periostin and Fibronectin were also higher in the 7.5% gelatin scaffold than in the 15% gelatin scaffold (P < 0.05). In conclusion, 7.5% gelatin scaffold is more beneficial to the proliferation and fibrogenic differentiation of hDPSCs.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Gelatin, Cross-Linking Reagents, Dental Pulp, Stem Cells, Tissue Engineering

CLC Number:

R394.2

Jiang Li-ming, Xia Shang, Song Ge, Chen Xu. Gelatin fibrous scaffolds promote fibrogenic differentiation of human dental pulp stem cells[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(13): 1981-1986.

Figures/Tables 2

2.1 明胶纤维支架的表面结构特点扫描电镜观察可见，7.5%明胶支架呈现纵横交错的纤维结构，纤维表面光滑，直径不均一，为(2.02 ±0.36) μm，45%的纤维直径分布于1.8-2.2 μm之间(图1A，图2A)。交联后的7.5%明胶纤维支架直径和孔隙均增大，纤维直径较为均一，为(3.15±0.52) μm，40%的纤维直径分布在3 μm左右(图1B，图2B)。交联前后的孔隙直径分别为(3.78±0.5) μm和(6.26±1.05) μm(图2C)。而15%明胶支架纤维出现纤维粘结，交联后支架表面纤维结构基本消失，几乎无孔隙，呈现平面结构(图1C和1D)。 2.2 明胶纤维支架的拉伸应力交联前，7.5%和15%明胶纤维支架材料的拉伸应力相似，为(0.143±0.019) MPa和(0.116±0.024) MPa，交联过程使两种支架材料的拉伸应力分别增加至(4.14±0.39) MPa和(1.893±0.096) MPa，7.5%明胶纤维支架的延展性优于15%明胶支架(t =3.899，P < 0.05)，见图2D。 2.3 人牙髓干细胞的培养和鉴定结果流式细胞术检测细胞表面标记物，结果显示CD73(98.8±0.15)%，CD90(91.4±0.35)%呈阳性表达，CD45(0.22±0.03)%、CD31(0.025±0.01)%呈阴性表达，见图3A。牙髓组织经酶消化后，原代培养的人牙髓干细胞贴壁生长，形态呈长梭形，类似成纤维细胞，见图3B。经成骨诱导培养21 d后，细胞可见明显的矿化结节形成，茜素红染色呈阳性，见图3C。经成脂诱导培养21 d后，可见脂滴形成，油红O染色呈阳性，见图3D。 2.4 明胶纤维支架对人牙髓干细胞增殖活性的影响明胶支架具有良好的细胞生物相容性，人牙髓干细胞可以在明胶纤维支架上黏附和生长。第1天时，两种支架材料上的细胞数量无明显差异，第3天时，细胞数量均增高，第7天时，7.5%明胶纤维支架上的细胞数量明显高于15%明胶支架组(P < 0.05)，表明7.5%明胶支架的多孔纤维结构更利于细胞的增殖，见图4。 2.5 明胶纤维支架促进人牙髓干细胞的成纤维分化 Collagen Ⅰ是牙髓组织中重要的细胞外基质，α-SMA、Fibronectin和Periostin均是纤维结缔组织标志性因子。第7天时，7.5%明胶纤维支架组的Collagen Ⅰ水平显著增高，为15%明胶纤维支架组的22.2倍，第14天时，7.5%明胶纤维支架组CollagenⅠ水平约为15%明胶纤维支架组的2倍，证明该支架的纳米纤维结构可以促进人牙髓干细胞向成纤维细胞分化。第7天时，7.5%明胶纤维支架组的α- SMA和Periostin水平分别是15%明胶纤维支架组的5.7倍和2.28倍，两组间Fibronectin的表达差异无显著性意义(P > 0.05)；第14天时，7.5%明胶纤维支架组α-SMA水平为15%明胶纤维支架组的1.6倍，Fibronectin的表达增高为15%明胶纤维支架组的2.24倍(P < 0.05)，两组Periostin的表达差异无显著性意义(P > 0.05)，见图5。"

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