Adhesion and proliferation of dental pulp stem cells on the chitosan-fibrin composite scaffold

doi:10.3969/j.issn.2095-4344.2017.10.013

Abstract

Abstract:

BACKGROUND: With the rapid development of tissue engineering, a single biological scaffold material is hard to meet the needs of tissue engineering. Therefore, composite scaffolds with excellent performance will be obtained by combining two or more kinds of materials.
OBJECTIVE: To detect the adherence and proliferation of dental pulp stem cells on the Chitosan-fibrin composite scaffold.
METHODS: Dental pulp stem cells were isolated and extracted from C57 neonatal rats through modified enzyme-digestion method, and subcultured to the third generation, followed by adipogenic and osteogenic induction in vitro. Then, induced cells were identified. The chitosan-fibrinogen composite scaffold was prepared, and the pore size and porosity were determined. The chitosan-fibrin composite scaffold was co-cultured with passage 3 dental pulp stem cells to observe the cell proliferation by MTT assay, and the morphology, cell adhesion, proliferation and extracellular matrix secretion were observed under scanning electron microscope, while cells were inoculated directly on the bottom of culture plate as controls.
RESULTS AND CONCLUSION: The dental pulp stem cells were successfully isolated and cultivated, and positive for osteogenic and adipogenic differentiation. The pore size and porosity of the composite scaffold was (105.32±22.10) μm and (87.714±1.276)%, respectively. The “S”-shaped proliferation curve in the experimental group was similar with that in the control group; the proliferation rate in the experimental group was significantly higher than that in the control group after 4-8 days of culture (P < 0.05). At the 2nd day after co-culture, the cells adhered tightly and grew well onto the composite scaffold; at the 4th day, the cells began to proliferate obviously full of extracellular matrix, and the cell volume was enlarged; the surface and pores of the scaffold were full of cells at the 6th day. These results indicate grew fast obviously that rich in began to proliferate obviously full of that the chitosan-fibrin composite scaffold is suitable for the adhesion and proliferation of dental pulp stem cells.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

Key words: Chitosan, Fibrinogen, Dental Pulp, Stem Cells, Tissue Engineering

CLC Number:

R318

Zheng Li-zhu, Li Xiao-bing, Zhang Miao, Yu Lu, Liu Yi-shan. Adhesion and proliferation of dental pulp stem cells on the chitosan-fibrin composite scaffold[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(10): 1552-1557.

Figures/Tables 5

References

[1]杨媛,彭楚,芳秦满,等.牙髓血运重建术治疗年轻恒牙根尖周病变的临床效果观察[J].中华口腔医学杂志,2013,48(2):81-85.

[2]李州,许庆安.干细胞和支架与牙髓再生及其血运重建[J].国际口腔医学杂志,2016,43(3):298-302.

[3]Wang Y, Zhao Y, Jia W, et al. Preliminary study on dental pulp stem cell-mediated pulp regeneration in canine immature permanent teeth. J Endod. 2013;39(2):195-201.

[4]D'Mello G, Moloney L. Management of coronal discolouration following a regenerative endodontic procedure in a maxillary incisor. Aust Dent J. 2016; 10(3):121-128.

[5]Bhardwaj N, Chakraborty S, Kundu SC. Freeze-gelled silk fibroin protein scaffolds for potential applications in soft tissue engineering. Int J Biol Macromol. 2011;49(3):260-267.

[6]Woloszyk A, Buschmann J, Waschkies C, et al. Human Dental Pulp Stem Cells and Gingival Fibroblasts Seeded into Silk Fibroin Scaffolds Have the Same Ability in Attracting Vessels. Front Physiol. 2016;7:140.

[7]雷鸣,高丽娜,陈发明,等.牙髓组织工程和再生中生物支架材料的进展[J].牙体牙髓牙周病学杂志,2013,23(1):51-56.

[8]Cao B, Yin J, Yan S, et al. Porous scaffolds based on cross-linking of poly(L-glutamic acid). Macromol Biosci. 2011; 11(3):427-434.

[9]别利克孜•卡德尔,刘奕杉,王璇,等.改良酶消化法分离培养人乳牙牙髓干细胞[J].中国组织工程研究,2013,17(10):1793-1800.

[10]Gong T, Heng BC, Lo EC, et al. Current Advance and Future Prospects of Tissue Engineering Approach to Dentin/Pulp Regenerative Therapy. Stem Cells Int. 2016;2016:9204574.

[11]Conde MC, Chisini LA, Demarco FF, et al. Stem cell-based pulp tissue engineering: variables enrolled in translation from the bench to the bedside, a systematic review of literature. Int Endod J. 2016;49(6):543-550.

[12]Piva E, Silva AF, Nör JE. Functionalized scaffolds to control dental pulp stem cell fate. J Endod. 2014;40(4 Suppl):S33-40.

[13]王洁,刘加强,房兵.牙髓干细胞在组织工程中的研究进展[J].口腔颌面修复学杂志,2013,14(3):183-186.

[14]史诗彧,谢家敏. 牙髓干细胞在组织工程领域的研究进展[J].华西口腔医学杂志,2015,33(6):656-659.

[15]任飞,周慧,刘建平,等.转化生长因子β3联合肝素在人乳牙牙髓干细胞向成牙本质样细胞分化中的作用[J].实用医学杂志,2014, 30(12):1887-1890.

[16]王亦菁,张晓东,李里,等.三维培养的牙髓干细胞参与牙髓损伤修复能力的实验研究[J].临床口腔医学杂志,2011,27(10): 582-584.

[17]贺慧霞,金岩,史俊南, 等. bFGF、TGF-β对人牙髓干细胞增殖和分化能力的影响[J].牙体牙髓牙周病学杂志,2004,14(7): 359-363.

[18]林颖,秦伟,邹瑞, 等.促丝裂原激活蛋白激酶在牙髓干细胞向成牙本质细胞分化和牙髓损伤修复中的作用[J].国际口腔医学杂志,2016,43(3):343-347.

[19]Leong DJ, Setzer FC, Trope M, et al. Biocompatibility of two experimental scaffolds for regenerative endodontics. Restor Dent Endod. 2016;41(2):98-105.

[20]年争好,李晖,李瑞欣,等.纳米羟基磷灰石/胶原蛋白/丝素蛋白复合骨组织工程支架材料的生物相容性[J].中国组织工程研究, 2015,19(8):1149-1154.

[21]吕孝帅,李正茂,王海燕, 等. 生物活性玻璃-丝素蛋白复合膜支持人牙髓干细胞增殖与分化初探[J].中华口腔医学杂志,2015, 50(12):725-730.

[22]李珊,樊明文,梁素霞.成人牙髓干细胞与壳聚糖-磷酸三钙复合材料相容性试验研究[J].口腔医学研究,2006,22(3):264-266.

[23]Baniasadi H, Ramazani S A A, Mashayekhan S. Fabrication and characterization of conductive chitosan/gelatin-based scaffolds for nerve tissue engineering. Int J Biol Macromol. 2015;74:360-366.

[24]Shukla SK, Mishra AK, Arotiba OA, et al. Chitosan-based nanomaterials: a state-of-the-art review. Int J Biol Macromol. 2013;59:46-58.

[25]Jridi M, Hajji S, Ayed HB, et al. Physical, structural, antioxidant and antimicrobial properties of gelatin-chitosan composite edible films. Int J Biol Macromol. 2014;67:373-379.

[26]Douglas TE, Krok-Borkowicz M, Macuda A, et al. Enrichment of thermosensitive chitosan hydrogels with glycerol and alkaline phosphatase for bone tissue engineering applications. Acta Bioeng Biomech. 2016;18(2):51-57.

[27]沈剑虹,杨甫,姚麒,等.结合bpV(pic)的壳聚糖支架对神经干细胞生长分化的影响[J].中华实验外科杂志,2015,32(8):1925-1928.

[28]薛震,牛丽媛,安刚,等.纳米羟基磷灰石/壳聚糖/半水硫酸钙为可注射骨组织工程支架材料的可行性[J].中国组织工程研究,2015, 19(8):1160-1164.

[29]祁伟,左强,崔维顶,等.采用纤维蛋白凝胶种植技术体外构建组织工程软骨[J].江苏医药,2011,37(12): 13 68-1370.

[30]Ding B, Gao H, Song J, et al. Tough and Cell-Compatible Chitosan Physical Hydrogels for Mouse Bone Mesenchymal Stem Cells in Vitro. ACS Appl Mater Interfaces. 2016;8(30): 19739-19746.

[31]刘清宇,王富友,刘俊利,等.含有天然钙化软骨层骨软骨支架的研制[J].中华创伤杂志,2014,30(5):467-470.

[32]鳄贤,邵林.骨组织工程多孔陶瓷支架制备工艺的研究进展[J].医学临床研究,2014,31(3):551-554.

[33]Buizer AT, Veldhuizen AG, Bulstra SK, et al. Static versus vacuum cell seeding on high and low porosity ceramic scaffolds. J Biomater Appl. 2014;29(1):3-13.

[34]聂姗姗,刘佳,张瑞涵,等.牙髓干细胞MHC分子表达与体外混合淋巴细胞的增殖[J].中国组织工程研究, 2014,18(50): 8162-8167.