鼻黏膜来源外胚间充质干细胞与磷酸钙骨水泥材料的体外相容性
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摘要
背景:利用磷酸钙骨水泥载间充质干细胞治疗骨缺损已取得令人满意的成果,但是鼻黏膜来源的外胚层间充质干细胞与磷酸钙骨水泥的体外相容性尚不清楚。目的:探讨磷酸钙骨水泥对大鼠鼻黏膜来源外胚间充质干细胞向成骨细胞分化的影响,进一步探讨鼻黏膜来源外胚间充质干细胞与磷酸钙骨水泥的生物相容性。方法:体外培养扩增鼻黏膜来源外胚间充质干细胞,并将其种植于磷酸钙骨水泥表面和培养板(对照组),均进行成骨诱导14 d。碱性磷酸酶活性法检测鼻黏膜来源外胚间充质干细胞的碱性磷酸酶活性,茜素红染色法测定钙结节面积大小,免疫荧光和免疫印迹法检测成骨诱导相关蛋白表达水平;鬼笔环肽染色法动态观察磷酸钙骨水泥表面的细胞形态;MTT法检测磷酸钙骨水泥对细胞增殖的影响。结果与结论:(1)成骨诱导14 d后,磷酸钙骨水泥表面的鼻黏膜来源外胚间充质干细胞碱性磷酸酶活性较高,形成的钙结节较大,骨相关蛋白表达水平较高;(2)磷酸钙骨水泥对鼻黏膜来源外胚间充质干细胞的增殖有一定的促进作用;(3)结果表明,鼻黏膜来源外胚间充质干细胞与磷酸钙骨水泥支架材料具有良好的生物相容性,磷酸钙骨水泥对鼻黏膜来源外胚间充质干细胞向成骨细胞分化表现出一定的促进作用。
BACKGROUND: The use of calcium phosphate cement(CPC) carrying mesenchymal stem cells for the treatment of bone defects has achieved satisfactory results. The in vitro compatibility of ectomesenchymal stem cells(EMSCs) derived from the nasal mucosa with CPC is unknown. OBJECTIVE: To investigate the effect of calcium phosphate cement on the differentiation of rat nasal mucosa-derived EMSCs into osteoblasts and to further explore the biocompatibility of EMSCs and CPC. METHODS: EMSCs were cultured, expanded in vitro and implanted onto the CPC surface or onto culture plates. Then, implanted EMSCs were induced to differentiate into osteoblasts for 14 days. Alkaline phosphatase activity assay was used to detect the alkaline phosphatase activity of EMSCs. The area of calcium nodules was determined by alizarin red staining. The expression level of osteogenesis-related proteins was detected by western blot assay and immunofluorescence method. Morphology of EMSCs on the CPC surface was dynamically observed using phalloidin staining. MTT assay was used to detect the proliferation of EMSCs on the CPC surface. RESULTS AND CONCLUSION: After 14 days of osteogenic induction, there was higher alkaline phosphatase activity, larger calcium nodules and higher levels of osteogenesis-related proteins in the EMSCs implanted on the surface of CPC than those implanted on the culture plates. The CPC had a certain role in promoting the proliferation of EMSCs. To conclude, EMSCs and CPC scaffolds have good biocompatibility, and moreover, CPC can certainly promote the differentiation of EMSCs into osteoblasts.
BACKGROUND: The use of calcium phosphate cement(CPC) carrying mesenchymal stem cells for the treatment of bone defects has achieved satisfactory results. The in vitro compatibility of ectomesenchymal stem cells(EMSCs) derived from the nasal mucosa with CPC is unknown. OBJECTIVE: To investigate the effect of calcium phosphate cement on the differentiation of rat nasal mucosa-derived EMSCs into osteoblasts and to further explore the biocompatibility of EMSCs and CPC. METHODS: EMSCs were cultured, expanded in vitro and implanted onto the CPC surface or onto culture plates. Then, implanted EMSCs were induced to differentiate into osteoblasts for 14 days. Alkaline phosphatase activity assay was used to detect the alkaline phosphatase activity of EMSCs. The area of calcium nodules was determined by alizarin red staining. The expression level of osteogenesis-related proteins was detected by western blot assay and immunofluorescence method. Morphology of EMSCs on the CPC surface was dynamically observed using phalloidin staining. MTT assay was used to detect the proliferation of EMSCs on the CPC surface. RESULTS AND CONCLUSION: After 14 days of osteogenic induction, there was higher alkaline phosphatase activity, larger calcium nodules and higher levels of osteogenesis-related proteins in the EMSCs implanted on the surface of CPC than those implanted on the culture plates. The CPC had a certain role in promoting the proliferation of EMSCs. To conclude, EMSCs and CPC scaffolds have good biocompatibility, and moreover, CPC can certainly promote the differentiation of EMSCs into osteoblasts.
引文
[1]王海波,杨新明,张瑛,等.大段骨缺损修复的组织工程学研究进展[J].中华生物医学工程杂志,2013,19(1):73-76.
[2]Kiernan CH, Wolvius EB, Brama PAJ, et al. The Immune Response to Allogeneic Differentiated Mesenchymal Stem Cells in the Context of Bone Tissue Engineering.Tissue Eng Part B Rev. 2018;24(1):75-83.
[3]王小志,何惠宇,杨楠,等.基因转染骨髓间充质干细胞复合同种异体骨修复绵羊极限骨缺损[J].中国组织工程研究, 2013,17(47):8141-8148.
[4]Smith BT, Santoro M, Grosfeld EC, et al. Incorporation of fast dissolving glucose porogens into an injectable calcium phosphate cement for bone tissue engineering. Acta Biomater. 2017;50:68-77.
[5]Chen Q, Zhou H, Hu P.Stemness distinctions between the ectomesenchymal stem cells from neonatal and adult mice. Acta Histochem. 2017;119(8):822-830.
[6]张志坚,刘锦波,姜平,等.大鼠嗅粘膜原代培养细胞的分类及细胞社会学特性研究[J].江苏大学学报(医学版),2004,14(1):4-7,96.
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[8]Delorme B, Nivet E, Gaillard J, et al. The human nose harbors a niche of olfactory ectomesenchymal stem cells displaying neurogenic and osteogenic properties.Stem Cells Dev. 2010;19(6):853-866.
[9]Chen Q, Zhang Z, Liu J, et al. A fibrin matrix promotes the differentiation of EMSCs isolated from nasal respiratory mucosa to myelinating phenotypical Schwann-like cells.Mol Cells. 2015;38(3):221-228.
[10]黄秋生,陆汉强,周月鹏,等.成人鼻黏膜间充质干细胞的体外培养和多向诱导分化[J].临床耳鼻咽喉头颈外科杂志, 2012,26(11):490-493,498.
[11]Shu X, Feng J, Feng J, et al. Combined delivery of bone morphogenetic protein-2 and insulin-like growth factor-1 from nano-poly(γ-glutamic acid)/β-tricalcium phosphate-based calcium phosphate cement and its effect on bone regeneration in vitro.J Biomater Appl. 2017;32(5):547-560.
[12]Darby I.Periodontal materials.Aust Dent J. 2011;56Suppl 1:107-118.
[13]温世锋,刘清华,郭奇峰,等.新型磷酸钙骨水泥与骨髓间充质干细胞的共培养研究[J].临床医学工程,2009,16(11):1-4.
[14]Gallinetti S, Mestres G, Canal C, et al. A novel strategy to enhance interfacial adhesion in fiber-reinforced calcium phosphate cement.J MechBehav Biomed Mater. 2017;75:495-503.
[15]Qiu G, Shi Z, Xu HHK, et al. Bone regeneration in minipigs via calcium phosphate cement scaffold delivering autologous bone marrow mesenchymal stem cells and platelet-rich plasma.J Tissue Eng Regen Med. 2018;12(2):e937-e948.
[16]Martin FT, Dwyer RM, Kelly J, et al. Potential role of mesenchymal stem cells(MSCs)in the breast tumour microenvironment:stimulation of epithelial to mesenchymal transition(EMT).Breast Cancer Res Treat.2010;124(2):317-326.
[17]Tohyama T, Lee VM, Rorke LB, et al. Nestin expression in embryonic human neuroepithelium and in human neuroepithelial tumor cells.Lab Invest. 1992;66(3):303-313.
[18]Richardson GD, Robson CN, Lang SH, et al. CD133, a novel marker for human prostatic epithelial stem cells.J Cell Sci. 2004;117(Pt 16):3539-3545.
[19]边兴艳.MTT比色法及其应用[J].国际检验医学杂志, 1998,19(2):83-85.
[20]Bessey OA, Lowry OH, Brock MJ.A method for the rapid determination of alkaline phosphates with five cubic millimeters of serum.J Biol Chem.1946;164:321-329.
[21]Kulikowski E, Halliday C, Johansson J, et al. Apabetalone Mediated Epigenetic Modulation is Associated with Favorable Kidney Function and Alkaline Phosphatase Profile in Patients with Chronic Kidney Disease.Kidney Blood Press Res. 2018;43(2):449-457.
[22]Tang J, Saito T.Elucidation on Predominant Pathways Involved in the Differentiation and Mineralization of Odontoblast-Like Cells by Selective Blockade of Mitogen-Activated Protein Kinases.Biomed Res Int. 2018;2018:2370438.
[23]Miller EJ. Collagen types:Structure, distribution, and functions.Collagen:CRC Press, 2018:139-156.
[24]McCully M, Conde J, Baptista P, et al. Nanoparticle-antagomiR based targeting of miR-31 to induce osterix and osteocalcin expression in mesenchymal stem cells.PLoS One. 2018;13(2):e0192562.
[25]Rickard DJ, Sullivan TA, Shenker BJ, et al. Induction of rapid osteoblast differentiation in rat bone marrow stromal cell cultures by dexamethasone and BMP-2.Dev Biol. 1994;161(1):218-228.
[26]Cha JK, Jung UW, Thoma DS, et al. Osteogenic efficacy of BMP-2mixed with hydrogel and bone substitute in peri-implant dehiscence defects in dogs:16 weeks of healing.Clin Oral Implants Res. 2018;29(3):300-308.
[27]Mu?oz-Pacheco JM, Zambrano-Serrano E, Félix-Beltrán O, et al.Synchronization of PWL function-based 2D and 3D multi-scroll chaotic systems. Nonlinear Dynamics. 2012;70(2):1633-1643.
[2]Kiernan CH, Wolvius EB, Brama PAJ, et al. The Immune Response to Allogeneic Differentiated Mesenchymal Stem Cells in the Context of Bone Tissue Engineering.Tissue Eng Part B Rev. 2018;24(1):75-83.
[3]王小志,何惠宇,杨楠,等.基因转染骨髓间充质干细胞复合同种异体骨修复绵羊极限骨缺损[J].中国组织工程研究, 2013,17(47):8141-8148.
[4]Smith BT, Santoro M, Grosfeld EC, et al. Incorporation of fast dissolving glucose porogens into an injectable calcium phosphate cement for bone tissue engineering. Acta Biomater. 2017;50:68-77.
[5]Chen Q, Zhou H, Hu P.Stemness distinctions between the ectomesenchymal stem cells from neonatal and adult mice. Acta Histochem. 2017;119(8):822-830.
[6]张志坚,刘锦波,姜平,等.大鼠嗅粘膜原代培养细胞的分类及细胞社会学特性研究[J].江苏大学学报(医学版),2004,14(1):4-7,96.
[7]Zhang Z, He Q, Deng W, et al. Nasal ectomesenchymal stem cells:multi-lineage differentiation and transformation effects on fibrin gels.Biomaterials. 2015;49:57-67.
[8]Delorme B, Nivet E, Gaillard J, et al. The human nose harbors a niche of olfactory ectomesenchymal stem cells displaying neurogenic and osteogenic properties.Stem Cells Dev. 2010;19(6):853-866.
[9]Chen Q, Zhang Z, Liu J, et al. A fibrin matrix promotes the differentiation of EMSCs isolated from nasal respiratory mucosa to myelinating phenotypical Schwann-like cells.Mol Cells. 2015;38(3):221-228.
[10]黄秋生,陆汉强,周月鹏,等.成人鼻黏膜间充质干细胞的体外培养和多向诱导分化[J].临床耳鼻咽喉头颈外科杂志, 2012,26(11):490-493,498.
[11]Shu X, Feng J, Feng J, et al. Combined delivery of bone morphogenetic protein-2 and insulin-like growth factor-1 from nano-poly(γ-glutamic acid)/β-tricalcium phosphate-based calcium phosphate cement and its effect on bone regeneration in vitro.J Biomater Appl. 2017;32(5):547-560.
[12]Darby I.Periodontal materials.Aust Dent J. 2011;56Suppl 1:107-118.
[13]温世锋,刘清华,郭奇峰,等.新型磷酸钙骨水泥与骨髓间充质干细胞的共培养研究[J].临床医学工程,2009,16(11):1-4.
[14]Gallinetti S, Mestres G, Canal C, et al. A novel strategy to enhance interfacial adhesion in fiber-reinforced calcium phosphate cement.J MechBehav Biomed Mater. 2017;75:495-503.
[15]Qiu G, Shi Z, Xu HHK, et al. Bone regeneration in minipigs via calcium phosphate cement scaffold delivering autologous bone marrow mesenchymal stem cells and platelet-rich plasma.J Tissue Eng Regen Med. 2018;12(2):e937-e948.
[16]Martin FT, Dwyer RM, Kelly J, et al. Potential role of mesenchymal stem cells(MSCs)in the breast tumour microenvironment:stimulation of epithelial to mesenchymal transition(EMT).Breast Cancer Res Treat.2010;124(2):317-326.
[17]Tohyama T, Lee VM, Rorke LB, et al. Nestin expression in embryonic human neuroepithelium and in human neuroepithelial tumor cells.Lab Invest. 1992;66(3):303-313.
[18]Richardson GD, Robson CN, Lang SH, et al. CD133, a novel marker for human prostatic epithelial stem cells.J Cell Sci. 2004;117(Pt 16):3539-3545.
[19]边兴艳.MTT比色法及其应用[J].国际检验医学杂志, 1998,19(2):83-85.
[20]Bessey OA, Lowry OH, Brock MJ.A method for the rapid determination of alkaline phosphates with five cubic millimeters of serum.J Biol Chem.1946;164:321-329.
[21]Kulikowski E, Halliday C, Johansson J, et al. Apabetalone Mediated Epigenetic Modulation is Associated with Favorable Kidney Function and Alkaline Phosphatase Profile in Patients with Chronic Kidney Disease.Kidney Blood Press Res. 2018;43(2):449-457.
[22]Tang J, Saito T.Elucidation on Predominant Pathways Involved in the Differentiation and Mineralization of Odontoblast-Like Cells by Selective Blockade of Mitogen-Activated Protein Kinases.Biomed Res Int. 2018;2018:2370438.
[23]Miller EJ. Collagen types:Structure, distribution, and functions.Collagen:CRC Press, 2018:139-156.
[24]McCully M, Conde J, Baptista P, et al. Nanoparticle-antagomiR based targeting of miR-31 to induce osterix and osteocalcin expression in mesenchymal stem cells.PLoS One. 2018;13(2):e0192562.
[25]Rickard DJ, Sullivan TA, Shenker BJ, et al. Induction of rapid osteoblast differentiation in rat bone marrow stromal cell cultures by dexamethasone and BMP-2.Dev Biol. 1994;161(1):218-228.
[26]Cha JK, Jung UW, Thoma DS, et al. Osteogenic efficacy of BMP-2mixed with hydrogel and bone substitute in peri-implant dehiscence defects in dogs:16 weeks of healing.Clin Oral Implants Res. 2018;29(3):300-308.
[27]Mu?oz-Pacheco JM, Zambrano-Serrano E, Félix-Beltrán O, et al.Synchronization of PWL function-based 2D and 3D multi-scroll chaotic systems. Nonlinear Dynamics. 2012;70(2):1633-1643.