载重组人血管内皮生长因子165的邻苯二酚壳聚糖促进乳牙牙髓干细胞血管形成的研究
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摘要
目的:体外研究载重组人血管内皮生长因子(recombinant human vascular endothelial growth factor,rhVEGF165)的邻苯二酚壳聚糖(catechol-chitosan,Cat-CS)支架材料对乳牙牙髓干细胞(stem cells from pulpof deciduous teeth,SHED)血管形成的影响,为其在年轻恒牙牙髓再生中的应用提供理论基础。方法:结合酶联合消化法和倒置贴壁法分离培养SHED。经细胞形态、碱性磷酸酶和茜素红染色对SHED进行鉴定,通过噻唑蓝(methyl thiazol tetrazolium,MTT)法检测和对比壳聚糖(chitosan,CS)及Cat-CS的生物相容性,HE染色及扫描电镜(scanning electron microscope,SEM)检测细胞在CS及Cat-CS上的粘附,实时定量聚合酶链反应(real-time quantitative polymerase chain reaction,real-time PCR)检测血管内皮细胞表面标记物CD31、血管内皮生长因子受体2(VEGFR2)、血管内皮钙粘蛋白(VE-cadherin)的表达。结果:SHED具有较强的增殖能力,碱性磷酸酶及茜素红染色阳性;MTT结果显示CS及Cat-CS对细胞无明显毒性;HE染色及SEM观察结果显示相比于CS,Cat-CS更有利于细胞在孔径内的延伸以及在其表面的粘附;与空白对照组比较,CS及Cat-CS能促进SHED表达CD31、VEGFR2和VE-cadherin,Cat-CS与rhVEGF165复合后,这些基因的表达进一步增加(P<0.05)。结论:Cat-CS具有良好的生物相容性,可以作为牙髓再生良好的支架材料。
Objective: To study the effects of rhVEGF165 loaded catechol chitosan(cat-cs) scaffold on the angiogenesis of stem cells from human exfoliated deciduous teeth(SHED) in young permanent teeth. Methods: SHED was isolated and cultured by enzyme digestion and inverted adherent method. The stemness of SHEDs was identified by cell morphology, alkaline phosphatase, and alizarin red staining. The biocompatibility of CS and Cat-CS was detected by MTT, HE staining, and scanning electron microscope(SEM). The expression of endothelial cell marker CD31, VEGFR2, and VE-cadherin was detected by real-time PCR. Results: SHED had strong reproductive activity. Alkaline phosphatase and alizarin red staining revealed differentiation of SHED. The MTT results showed no obvious toxicity of Cat-CS to SHED. HE staining and SEM showed Cat-CS was more favorable for cell extension and adhesion. Cat-CS could efficiently promote more expression of CD31, VEGFR2, and VE-cadherin than CS. And the expression of these genes is further promoted when Cat-CS is combined with rhVEGF165(P<0.05). Conclusion: Cat-CS has good biocompatibility and can be used as a good scaffold for pulp regeneration.
Objective: To study the effects of rhVEGF165 loaded catechol chitosan(cat-cs) scaffold on the angiogenesis of stem cells from human exfoliated deciduous teeth(SHED) in young permanent teeth. Methods: SHED was isolated and cultured by enzyme digestion and inverted adherent method. The stemness of SHEDs was identified by cell morphology, alkaline phosphatase, and alizarin red staining. The biocompatibility of CS and Cat-CS was detected by MTT, HE staining, and scanning electron microscope(SEM). The expression of endothelial cell marker CD31, VEGFR2, and VE-cadherin was detected by real-time PCR. Results: SHED had strong reproductive activity. Alkaline phosphatase and alizarin red staining revealed differentiation of SHED. The MTT results showed no obvious toxicity of Cat-CS to SHED. HE staining and SEM showed Cat-CS was more favorable for cell extension and adhesion. Cat-CS could efficiently promote more expression of CD31, VEGFR2, and VE-cadherin than CS. And the expression of these genes is further promoted when Cat-CS is combined with rhVEGF165(P<0.05). Conclusion: Cat-CS has good biocompatibility and can be used as a good scaffold for pulp regeneration.
引文
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[13] 赵易尔,金丹.壳聚糖复合材料在骨组织工程中的应用[J].基因组学与应用生物学,2015,34(4):842-848.
[14] Ryu JH, Hong S, Lee H. Bio-inspired adhesive catechol-conjugated chitosan for biomedical applications: A mini review [J]. Acta Biomaterialia, 2015, 27:101-15.
[15] 刘伟,凌均棨.牙髓干细胞诱导分化为血管内皮细胞的研究进展[J].国际口腔医学杂志,2014,41(6):707-711.
[2] Li X, Chi M, Xie X, et al. Pulp regeneration in a full-length human tooth root using a hierarchical nanofibrous microsphere system [J]. Acta Biomaterialia, 2016, 35:57-67.
[3] Rouwkema J, Khademhosseini A. Vascularization and angiogenesis in tissue engineering: beyond creating static networks [J]. Trends Biotechnol, 2016, 34(9):733-745.
[4] Gaihre B, Jayasuriya AC, et al. Comparative investigation of porous nano-hydroxyapaptite/chitosan,nano-zirconia/chitosan and novel nano-calcium zirconate/chitosan composite scaffolds for their potential applications in bone regeneration [J]. Mater Sci Eng C Mater Biol Appl, 2018,91:330-339.
[5] Wu SW, Liu X, Miller NA, et al. Strengthening injectable thermo-sensitive NIPAAm-g-chitosan hydrogels using chemical cross-linking of disulfide bonds as scaffolds for tissue engineering [J]. Carbohydr Polym, 2018,192:308-316.
[6] Jiang T, Deng M, James R, et al. Micro- and nanofabrication of chitosan structures for regenerative engineering [J]. Acta Biomaterialia, 2014, 10(4):1632-1645.
[7] Xu J, Strandman S, Zhu JX, et al. Genipin-crosslinked catechol-chitosan mucoadhesive hydrogels for buccal drug delivery [J]. Biomaterials, 2015, 37:395-404.
[8] 杜欧,盖丽婷,王民艳,等.聚乳酸/羟基磷灰石复合材料的机械性能及其细胞毒性[J].吉林大学学报(医学版), 2014, 40(2):300-305.
[9] Liu M, Zeng X, Ma C, et al. Injectable hydrogels for cartilage and bone tissue engineering [J]. Bone Res, 2017, 5:75-94.
[10] 凌均棨,毛剑.牙髓再生的研究现状与发展前景[J].中华口腔医学杂志,2018,53(6):361-366.
[11] Sakai VT, Zhang Z, Dong Z, et al. SHED differentiate into functional odontoblasts and endothelium [J]. J Dent Res, 2010, 89(8):791-796.
[12] 刘飞,刘阳,戴姗姗,等.CM-DiI体外标记人乳牙牙髓干细胞的可行性研究[J].口腔医学研究,2016,32(3):290-293.
[13] 赵易尔,金丹.壳聚糖复合材料在骨组织工程中的应用[J].基因组学与应用生物学,2015,34(4):842-848.
[14] Ryu JH, Hong S, Lee H. Bio-inspired adhesive catechol-conjugated chitosan for biomedical applications: A mini review [J]. Acta Biomaterialia, 2015, 27:101-15.
[15] 刘伟,凌均棨.牙髓干细胞诱导分化为血管内皮细胞的研究进展[J].国际口腔医学杂志,2014,41(6):707-711.