Stem cells from human exfoliated teeth combined with fibrin facilitate bone regeneration in a rat periodontal bone defect model

doi:10.3969/j.issn.2095-4344.1660

Abstract

Abstract:

BACKGROUND: Oral and maxillofacial organs are important organs in the human body. Periodontal bone defects caused by infection and trauma are one of the common oral and maxillofacial diseases. The rapid development of tissue engineering in recent years has brought hope to the treatment of periodontal bone defects.
OBJECTIVE: To explore the repairing ability of stem cells from human exfoliated teeth (SHEDs) combined with fibrin in a rat periodontal bone defect model.
METHODS: We isolated and cultured SHEDs, and then induced these cells to differentiate into osteogenic cells in vitro for 2 weeks. Seventy-two Sprague-Dawley rats (purchased from the Nanjing Biomedical Research Institute of Nanjing University in China) were used to generate the periodontal bone defect model. The bone defect of 5 mm×4 mm×1 mm (length×width×depth) was made at the lower edge of the molar. These rat models were randomly divided into three groups, and PBS, fibrin and SHEDs+fibrin were implanted into the defect area. Animals were sacrificed at 1, 2, 4, 6 weeks after surgery, and the periodontal samples were isolated and analyzed histomorphologically.
RESULTS AND CONCLUSION: SHEDs had substantial osteogenic ability and western blot analysis showed that the expression of Runx2 and Alp was increased prominently after osteogenic induction in vitro. Hematoxylin-eosin staining showed that the percentage of newly formed bone in the SHEDs+fibrin group was significantly greater than that in the fribrin and PBS groups respectively at 2 and 4 weeks after transplantation. Immunohistochemical findings revealed that the expression of Runx2, Alp, Ocn in the SHEDs+fibrin group was improved significantly as compared with the fibrin and PBS groups. In conclusion, the transplantation of SHEDs promotes the repair and regeneration of the periodontal bone defect in rats, providing experimental evidence for human periodontal tissue repair with tissue engineering methods.

Key words: Tooth, Deciduous, Dental Pulp, Stem Cells, Fibrin, Tissue Engineering

CLC Number:

Xu Li, Song Hao, Kang Jie, Jia Guotao, Chen Chao, Wang Wei, Wang Yan, Zhang Nan, Han Fabin. Stem cells from human exfoliated teeth combined with fibrin facilitate bone regeneration in a rat periodontal bone defect model[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(13): 2061-2066.

Figures/Tables 2

2.1 脱落乳牙牙髓干细胞形态和成骨诱导活性从儿童乳牙中分离出牙髓干细胞并诱导分化为成骨细胞。茜素红染色结果显示牙髓干细胞在成骨诱导2周后能够形成明显的钙化结节。蛋白免疫印迹显示，Runx2、Alp蛋白表达分别比未处理对照组增加8.8倍和4.5倍，见图1。 2.2 实验动物数量分析 72只雄性SD大鼠均进入结果分析。 2.3 牙周骨缺损模型制备和乳牙牙髓干细胞移植在牙周骨上构建了5 mm×4 mm×1 mm(长×宽×深)的缺损。将15 μL纤维蛋白原溶液和含有2×106个牙髓干细胞的25 μL凝血酶溶液同时注射到缺损区域，约1 min后，液体变成凝胶，缝合肌肉层和皮肤层，见图2。 2.4 新骨组织形态学分析为了检测牙周骨缺损的新骨再生，术后1，2，4，6周获得大鼠牙周骨。用苏木精-伊红染色和组织形态学测量法分析大鼠骨缺损区域，见图3。第1周时，3组新骨形成百分比差异无显著性意义；第2，4周时，乳牙牙髓干细胞-纤维蛋白复合物组新骨形成百分比显著高于PBS组、纤维蛋白组；第6周时，各组愈合程度都比较好，新骨形成百分比差异无显著性意义。 2.5 通过Runx2，Alp和Ocn免疫组织化学染色评价乳牙牙髓干细胞对骨再生的作用在第1周和第2周，与PBS组、纤维蛋白组相比，乳牙牙髓干细胞-纤维蛋白复合物组Runx2阳性表达显著增强(P < 0.01)，见图4。在第4周，Runx2阳性表达显著下降，3组间差异无显著性意义。Runx2是成骨早期标志物，在第6周各组阳性细胞表达量很少。在第1周和第4周，与PBS组、纤维蛋白组相比，乳牙牙髓干细胞-纤维蛋白复合物组中Alp阳性表达显著增强 (P < 0.05)，见图5。在第2周，乳牙牙髓干细胞-纤维蛋白复合物组的Alp表达高于PBS组，但乳牙牙髓干细胞-纤维蛋白复合物组和纤维蛋白组之间差异无显著性意义。Alp是成骨早期标志物，在第6周各组阳性细胞表达量很少。随着时间的推移，成骨标记物Ocn的表达水平逐渐升高。在第2周时，3组间差异无显著性意义(P > 0.05)，在第4周和第6周，乳牙牙髓干细胞-纤维蛋白复合物组的Ocn表达显著高于其他2组(P < 0.05)，见图6。Ocn是成骨晚期标志物，在第1周时各组阳性细胞表达量很少。"

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