<i>In vitro</i>differentiation of human amniotic mesenchymal stem cells into ligament fibroblasts after induced by transforming growth factor β<sub>1</sub> and vascular endothelial growth factor _Chinese Journal of Reparative and Reconstructive Surgery

Authors：

ZOU Gang , LI Yuwan ^△ , JIN Ying , ZHU Xizhong , YANG Jibin , WANG Shengmin , YOU Qi , XIONG Huazhang ,  LIU Yi

The First Department of Orthopaedics, the Affiliated Hospital of Zunyi Medical College, Zunyi Guizhou, 563000, P.R.China;

Corresponding author：

LIU Yi, Email: 13308529536@163.com

Keywords：

Human amniotic mesenchymal stem cells; ligament fibroblasts; transforming growth factor β₁; vascular endothelial growth factor; tissue engineered ligament

DOI：

10.7507/1002-1892.201612090

Video：

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Objective To investigate whether human amniotic mesenchymal stem cells (hAMSCs) have the characteristics of mesenchymal stem cells (MSCs) and the differentiation capacity into ligament fibroblastsin vitro. Methods The hAMSCs were separated through trypsin and collagenase digestion from placenta, the phenotypic characteristics of hAMSCs were detected by flow cytometry, the cytokeratin-19 (CK-19) and vimentin expression of hAMSCs were tested through immunofluorescence staining. The hAMSCs at the 3rd passage were cultured with L-DMEM/F12 medium containing transforming growth factor β₁ (TGF-β₁) and vascular endothelial growth factor (VEGF) as the experimental group and with single L-DMEM/F12 medium as the control group. The morphology of hAMSCs was observed by inverted phase contrast microscope; the cellular activities and ability of proliferation were examined by cell counting kit-8 (CCK-8) method; the ligament fibroblasts related protein expressions including collagen type I, collagen type III, Fibronectin, and Tenascin-C were detected by immunofluorescence staining; specific mRNA expressions of ligament fibroblasts and angiogenesis including collagen type I, collagen type III, Fibronectin, α-smooth muscle actin (α-SMA), and VEGF were measured by real-time fluorescence quantitative PCR. Results The hAMSCs presented monolayer and adherent growth under inverted phase contrast microscope; the flow cytometry results demonstrated that hAMSCs expressed the MSCs phenotypes; the immunofluorescence staining results indicated the hAMSCs had high expression of the vimentin and low expression of CK-19; the hAMSCs possessed the differentiation ability into the osteoblasts, chondroblasts, and lipoblasts. The CCK-8 results displayed that cells reached the peak of growth curve at 7 days in each group, and the proliferation ability in the experimental group was significantly higher than that in the control group at 7 days (P<0.05). The immunofluorescence staining results showed that the expressions of collagen type I, collagen type III, Fibronectin, and Tenascin-C in the experimental group were significantly higher than those in the control group at 5, 10, and15 days after culture (P<0.05). The real-time fluorescence quantitative PCR results revealed that the mRNA relative expressions had an increasing tendency at varying degrees with time in the experimental group (P<0.05). The relative mRNA expressions of collagen type I, collagen type III, Fibronectin, α-SMA, and VEGF in the experimental group were significantly higher than those in the control group at the other time points (P<0.05), but no significant difference was found in the relative mRNA expressions of collagen type I, collagen type III, and VEGF between 2 groups at 5 days (P>0.05). Conclusion The hAMSCs possesses the characteristics of MSCs and good proliferation ability which could be chosen as seed cell source in tissue engineering. The expressions of ligament fibroblasts and angiogenesis related genes could be up-regulated, after inductionin vitro, and the synthesis of ligament fibroblasts related proteins could be strengthened. In addition, the application of TGF-β₁ and VEGF could be used as growth factors sources in constructing tissue engineered ligament.

Citation： ZOU Gang, LI Yuwan, JIN Ying, ZHU Xizhong, YANG Jibin, WANG Shengmin, YOU Qi, XIONG Huazhang, LIU Yi. In vitrodifferentiation of human amniotic mesenchymal stem cells into ligament fibroblasts after induced by transforming growth factor β₁ and vascular endothelial growth factor . Chinese Journal of Reparative and Reconstructive Surgery, 2017, 31(5): 582-593. doi: 10.7507/1002-1892.201612090 Copy

1.	张承昊, 金瑛, 范青洪, 等. rBMSC 和 rLFs 做为韧带组织工程种子细胞的特性比较. 遵义医学院学报, 2015, 38(1): 54-59.
2.	Smith SE, White RA, Grant DA, et al. Gold and Hydroxyapatite Nano-Composite Scaffolds for Anterior Cruciate Ligament Reconstruction:In Vitro Characterization. J Nanosci Nanotechnol, 2016, 16(1): 1160-1169.
3.	Zhi Y, Liu W, Zhang P, et al. Erratum to: Electrospun silk fibroin mat enhances tendon-bone healing in a rabbit extra-articular model. Biotechnol Letters, 2016, 38(10): 1837-1838.
4.	Cooper JA Jr, Bailey LO, Carter JN, et al. Evaluation of the anterior cruciate ligament, medial collateral ligament, achilles tendon and patellar tendon as cell sources for tissue-engineered ligament. Biomaterials, 2006, 27(13): 2747-2754.
5.	Leroy A, Nottelet B, Bony C, et al. PLA-poloxamer/poloxamine copolymers for ligament tissue engineering: sound macromolecular design for degradable scaffolds and MSC differentiation. Biomater Sci, 2015, 3(4): 617-626.
6.	付维力, 陈刚, 唐新, 等. BMP-12 重组腺病毒载体转染外周血 MSCs 向肌腱/韧带细胞分化研究. 中国修复重建外科杂志, 2015, 29(4): 472-476.
7.	Caplan AI. New era of cell-based orthopedic therapies. Tissue Eng Part B Rev, 2009, 15(2): 195-200.
8.	Maidhof R, Rafiuddin A, Chowdhury F, et al. Timing of mesenchymal stem cell delivery impacts the fate and therapeutic potential in intervertebral disc repair. J Orthop Res, 2017, 35(1): 32-40.
9.	Nogami M, Kimura T, Seki S, et al. A Human Amnion-Derived Extracellular Matrix-Coated Cell-Free Scaffold for Cartilage Repair:In Vitro andIn Vivo Studies. Tissue Eng Part A, 2016, 22(7-8): 680-688.
10.	洪佳琼, 高雅, 宋洁, 等. 人羊膜间充质干细胞与骨髓间充质干细胞的生物学特性及免疫抑制作用的比较. 中国实验血液学杂志, 2016, 24(3): 858-864.
11.	Kim SH, Bang SH, Kang SY, et al. Human amniotic membrane-derived stromal cells (hAMSC) interact depending on breast cancer cell type through secreted molecules. Tissue Cell, 2015, 47(1): 10-16.
12.	Cortes Y, Ojeda M, Araya D, et al. Isolation and multilineage differentiation of bone marrow mesenchymal stem cells from abattoir-derived bovine fetuses. BMC Vet Res, 2013, 9: 133.
13.	Vargas D, Rosales W, Lizcano F. Modifications of Human Subcutaneous ADMSC after PPARgamma Activation and Cold Exposition. Stem Cells Int, 2015, 2015: 196348.
14.	Fu Q, Tang NN, Zhang Q, et al. Preclinical Study of Cell Therapy for Osteonecrosis of the Femoral Head with Allogenic Peripheral Blood-Derived Mesenchymal Stem Cells. Yonsei Med J, 2016, 57(4): 1006-1015.
15.	金瑛, 李豫皖, 张承昊, 等. 体外诱导人羊膜间充质干细胞向韧带细胞分化的实验研究. 中国修复重建外科杂志, 2016, 30(2): 237-244.
16.	Filioli Uranio M, Dell’Aquila ME, Caira M, et al. Characterization andin vitro differentiation potency of early-passage canine amnion- and umbilical cord-derived mesenchymal stem cells as related to gestational age. Mol Reprod Dev, 2014, 81(6): 539-551.
17.	丁志, 杨松林. 间充质干细胞生物学特性及其分化潜能. 中国组织工程研究与临床康复, 2011, 15(1): 147-150.
18.	Ghebes CA, van Lente J, Post JN, et al. High-Throughput Screening Assay Identifies Small Molecules Capable of Modulating the BMP-2 and TGF-beta1 Signaling Pathway. SLAS Discov, 2017, 22(1): 40-50.
19.	Lee SB, Lim AR, Rah DK, et al. Modulation of heat shock protein 90 affects TGF-beta-induced collagen synthesis in human dermal fibroblast cells. Tissue Cell, 2016, 48(6): 616-623.
20.	Sasaki K, Kuroda R, Ishida K, et al. Enhancement of tendon-bone osteointegration of anterior cruciate ligament graft using granulocyte colony-stimulating factor. Am J Sports Med, 2008, 36(8): 1519-1527.
21.	Bissell L, Tibrewal S, Sahni V, et al. Growth factors and platelet rich plasma in anterior cruciate ligament reconstruction. Curr Stem Cell Res Ther, 2015, 10(1): 19-25.
22.	刘毅, 张承昊, 范青洪, 等. TGF-β₁ 和 bFGF 对单层共培养 BMSCs 和韧带成纤维细胞韧带特异性的影响. 中国修复重建外科杂志, 2014, 28(11): 1406-1412.
23.	王佃亮. 种子细胞——组织工程连载之三. 中国生物工程杂志, 2014, 34(7): 108-113.
24.	Montali M, Barachini S, Panvini FM, et al. Growth Factor Content in Human Sera Affects the Isolation of Mesangiogenic Progenitor Cells (MPCs) from Human Bone Marrow. Front Cell Dev Biol, 2016, 4: 114.
25.	Kalathur M, Baiguera S, Macchiarini P. Translating tissue-engineered tracheal replacement from bench to bedside. Cell Mol Life Sci, 2010, 67(24): 4185-4196.
26.	Niwa H, Masui S, Chambers I, et al. Phenotypic complementation establishes requirements for specific POU domain and generic transactivation function of Oct-3/4 in embryonic stem cells. Mol Cell Biol, 2002, 22(5): 1526-1536.
27.	Chehelcheraghi F, Eimani H, Homayoonsadraie S, et al. Effects of Acellular Amniotic Membrane Matrix and Bone Marrow-Derived Mesenchymal Stem Cells in Improving Random Skin Flap Survival in Rats. IranRed Crescent Med J, 2016, 18(6): e25588.
28.	方宁, 张路, 宋秀军, 等. 人羊膜间充质干细胞的分离、培养及鉴定. 遵义医学院学报, 2009, 32(3): 234-236.
29.	Bonomi A, Silini A, Vertua E, et al. Human amniotic mesenchymal stromal cells (hAMSCs) as potential vehicles for drug delivery in cancer therapy: anin vitro study. Stem Cell Res Ther, 2015, 6: 155.
30.	Kostjuk S, Loseva P, Chvartatskaya O, et al. Extracellular GC-rich DNA activates TLR9- and NF-kB-dependent signaling pathways in human adipose-derived mesenchymal stem cells (haMSCs). Expert Opin Biol Ther, 2012, 12 Suppl 1: S99-111.
31.	Miranda-Sayago JM, Fernandez-Arcas N, Reyes-Engel A, et al. Changes in CDKN2D, TP53, and miR125a expression: potential role in the evaluation of human amniotic fluid-derived mesenchymal stromal cell fitness. Genes Cells, 2012, 17(8): 673-687.
32.	Rutka JT. Expression of Concern: Dual regeneration of muscle and nerve by intravenous administration of human amniotic fluid-derived mesenchymal stem cells regulated by stromal cell-derived factor-1alpha in a sciatic nerve injury model. J Neurosurg, 2015, 123(6): 1605.
33.	Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy, 2006, 8(4): 315-317.
34.	Crane JL, Xian L, Cao X. Role of TGF-beta Signaling in Coupling Bone Remodeling. Methods Mol Biol, 2016, 1344: 287-300.
35.	Sulaiman W, Nguyen DH. Transforming growth factor beta 1, a cytokine with regenerative functions. Neural Regen Res, 2016, 11(10): 1549-1552.
36.	Utsunomiya T, Ishibazawa A, Nagaoka T, et al. Transforming Growth Factor-beta Signaling Cascade Induced by Mechanical Stimulation of Fluid Shear Stress in Cultured Corneal Epithelial Cells. Invest Ophthalmol Vis Sci, 2016, 57(14): 6382-6388.
37.	Chou PH, Wang ST, Ma HL, et al. Development of a two-step protocol for culture expansion of human annulus fibrosus cells with TGF-beta1 and FGF-2. Stem Cell Res Ther, 2016, 7(1): 89.
38.	Hara T, Yoshida E, Shinkai Y, et al. Biglycan Intensifies ALK5-Smad2/3 Signaling by TGF-beta1 and Downregulates Syndecan-4 in Cultured Vascular Endothelial Cells. J Cell Biochem, 2017, 118(5): 1087-1096.
39.	Farhat YM, Al-Maliki AA, Easa A, et al. TGF-beta1 Suppresses Plasmin and MMP Activity in Flexor Tendon Cells via PAI-1: Implications for Scarless Flexor Tendon Repair. J Cell Physiol, 2015, 230(2): 318-326.
40.	Chen J, Yang L, Guo L, et al. Sodium hyaluronate as a drug-release system for VEGF 165 improves graft revascularization in anterior cruciate ligament reconstruction in a rabbit model. Exp Ther Med, 2012, 4(3): 430-434.

1. 张承昊, 金瑛, 范青洪, 等. rBMSC 和 rLFs 做为韧带组织工程种子细胞的特性比较. 遵义医学院学报, 2015, 38(1): 54-59.
2. Smith SE, White RA, Grant DA, et al. Gold and Hydroxyapatite Nano-Composite Scaffolds for Anterior Cruciate Ligament Reconstruction:In Vitro Characterization. J Nanosci Nanotechnol, 2016, 16(1): 1160-1169.
3. Zhi Y, Liu W, Zhang P, et al. Erratum to: Electrospun silk fibroin mat enhances tendon-bone healing in a rabbit extra-articular model. Biotechnol Letters, 2016, 38(10): 1837-1838.
4. Cooper JA Jr, Bailey LO, Carter JN, et al. Evaluation of the anterior cruciate ligament, medial collateral ligament, achilles tendon and patellar tendon as cell sources for tissue-engineered ligament. Biomaterials, 2006, 27(13): 2747-2754.
5. Leroy A, Nottelet B, Bony C, et al. PLA-poloxamer/poloxamine copolymers for ligament tissue engineering: sound macromolecular design for degradable scaffolds and MSC differentiation. Biomater Sci, 2015, 3(4): 617-626.
6. 付维力, 陈刚, 唐新, 等. BMP-12 重组腺病毒载体转染外周血 MSCs 向肌腱/韧带细胞分化研究. 中国修复重建外科杂志, 2015, 29(4): 472-476.
7. Caplan AI. New era of cell-based orthopedic therapies. Tissue Eng Part B Rev, 2009, 15(2): 195-200.
8. Maidhof R, Rafiuddin A, Chowdhury F, et al. Timing of mesenchymal stem cell delivery impacts the fate and therapeutic potential in intervertebral disc repair. J Orthop Res, 2017, 35(1): 32-40.
9. Nogami M, Kimura T, Seki S, et al. A Human Amnion-Derived Extracellular Matrix-Coated Cell-Free Scaffold for Cartilage Repair:In Vitro andIn Vivo Studies. Tissue Eng Part A, 2016, 22(7-8): 680-688.
10. 洪佳琼, 高雅, 宋洁, 等. 人羊膜间充质干细胞与骨髓间充质干细胞的生物学特性及免疫抑制作用的比较. 中国实验血液学杂志, 2016, 24(3): 858-864.
11. Kim SH, Bang SH, Kang SY, et al. Human amniotic membrane-derived stromal cells (hAMSC) interact depending on breast cancer cell type through secreted molecules. Tissue Cell, 2015, 47(1): 10-16.
12. Cortes Y, Ojeda M, Araya D, et al. Isolation and multilineage differentiation of bone marrow mesenchymal stem cells from abattoir-derived bovine fetuses. BMC Vet Res, 2013, 9: 133.
13. Vargas D, Rosales W, Lizcano F. Modifications of Human Subcutaneous ADMSC after PPARgamma Activation and Cold Exposition. Stem Cells Int, 2015, 2015: 196348.
14. Fu Q, Tang NN, Zhang Q, et al. Preclinical Study of Cell Therapy for Osteonecrosis of the Femoral Head with Allogenic Peripheral Blood-Derived Mesenchymal Stem Cells. Yonsei Med J, 2016, 57(4): 1006-1015.
15. 金瑛, 李豫皖, 张承昊, 等. 体外诱导人羊膜间充质干细胞向韧带细胞分化的实验研究. 中国修复重建外科杂志, 2016, 30(2): 237-244.
16. Filioli Uranio M, Dell’Aquila ME, Caira M, et al. Characterization andin vitro differentiation potency of early-passage canine amnion- and umbilical cord-derived mesenchymal stem cells as related to gestational age. Mol Reprod Dev, 2014, 81(6): 539-551.
17. 丁志, 杨松林. 间充质干细胞生物学特性及其分化潜能. 中国组织工程研究与临床康复, 2011, 15(1): 147-150.
18. Ghebes CA, van Lente J, Post JN, et al. High-Throughput Screening Assay Identifies Small Molecules Capable of Modulating the BMP-2 and TGF-beta1 Signaling Pathway. SLAS Discov, 2017, 22(1): 40-50.
19. Lee SB, Lim AR, Rah DK, et al. Modulation of heat shock protein 90 affects TGF-beta-induced collagen synthesis in human dermal fibroblast cells. Tissue Cell, 2016, 48(6): 616-623.
20. Sasaki K, Kuroda R, Ishida K, et al. Enhancement of tendon-bone osteointegration of anterior cruciate ligament graft using granulocyte colony-stimulating factor. Am J Sports Med, 2008, 36(8): 1519-1527.
21. Bissell L, Tibrewal S, Sahni V, et al. Growth factors and platelet rich plasma in anterior cruciate ligament reconstruction. Curr Stem Cell Res Ther, 2015, 10(1): 19-25.
22. 刘毅, 张承昊, 范青洪, 等. TGF-β₁ 和 bFGF 对单层共培养 BMSCs 和韧带成纤维细胞韧带特异性的影响. 中国修复重建外科杂志, 2014, 28(11): 1406-1412.
23. 王佃亮. 种子细胞——组织工程连载之三. 中国生物工程杂志, 2014, 34(7): 108-113.
24. Montali M, Barachini S, Panvini FM, et al. Growth Factor Content in Human Sera Affects the Isolation of Mesangiogenic Progenitor Cells (MPCs) from Human Bone Marrow. Front Cell Dev Biol, 2016, 4: 114.
25. Kalathur M, Baiguera S, Macchiarini P. Translating tissue-engineered tracheal replacement from bench to bedside. Cell Mol Life Sci, 2010, 67(24): 4185-4196.
26. Niwa H, Masui S, Chambers I, et al. Phenotypic complementation establishes requirements for specific POU domain and generic transactivation function of Oct-3/4 in embryonic stem cells. Mol Cell Biol, 2002, 22(5): 1526-1536.
27. Chehelcheraghi F, Eimani H, Homayoonsadraie S, et al. Effects of Acellular Amniotic Membrane Matrix and Bone Marrow-Derived Mesenchymal Stem Cells in Improving Random Skin Flap Survival in Rats. IranRed Crescent Med J, 2016, 18(6): e25588.
28. 方宁, 张路, 宋秀军, 等. 人羊膜间充质干细胞的分离、培养及鉴定. 遵义医学院学报, 2009, 32(3): 234-236.
29. Bonomi A, Silini A, Vertua E, et al. Human amniotic mesenchymal stromal cells (hAMSCs) as potential vehicles for drug delivery in cancer therapy: anin vitro study. Stem Cell Res Ther, 2015, 6: 155.
30. Kostjuk S, Loseva P, Chvartatskaya O, et al. Extracellular GC-rich DNA activates TLR9- and NF-kB-dependent signaling pathways in human adipose-derived mesenchymal stem cells (haMSCs). Expert Opin Biol Ther, 2012, 12 Suppl 1: S99-111.
31. Miranda-Sayago JM, Fernandez-Arcas N, Reyes-Engel A, et al. Changes in CDKN2D, TP53, and miR125a expression: potential role in the evaluation of human amniotic fluid-derived mesenchymal stromal cell fitness. Genes Cells, 2012, 17(8): 673-687.
32. Rutka JT. Expression of Concern: Dual regeneration of muscle and nerve by intravenous administration of human amniotic fluid-derived mesenchymal stem cells regulated by stromal cell-derived factor-1alpha in a sciatic nerve injury model. J Neurosurg, 2015, 123(6): 1605.
33. Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy, 2006, 8(4): 315-317.
34. Crane JL, Xian L, Cao X. Role of TGF-beta Signaling in Coupling Bone Remodeling. Methods Mol Biol, 2016, 1344: 287-300.
35. Sulaiman W, Nguyen DH. Transforming growth factor beta 1, a cytokine with regenerative functions. Neural Regen Res, 2016, 11(10): 1549-1552.
36. Utsunomiya T, Ishibazawa A, Nagaoka T, et al. Transforming Growth Factor-beta Signaling Cascade Induced by Mechanical Stimulation of Fluid Shear Stress in Cultured Corneal Epithelial Cells. Invest Ophthalmol Vis Sci, 2016, 57(14): 6382-6388.
37. Chou PH, Wang ST, Ma HL, et al. Development of a two-step protocol for culture expansion of human annulus fibrosus cells with TGF-beta1 and FGF-2. Stem Cell Res Ther, 2016, 7(1): 89.
38. Hara T, Yoshida E, Shinkai Y, et al. Biglycan Intensifies ALK5-Smad2/3 Signaling by TGF-beta1 and Downregulates Syndecan-4 in Cultured Vascular Endothelial Cells. J Cell Biochem, 2017, 118(5): 1087-1096.
39. Farhat YM, Al-Maliki AA, Easa A, et al. TGF-beta1 Suppresses Plasmin and MMP Activity in Flexor Tendon Cells via PAI-1: Implications for Scarless Flexor Tendon Repair. J Cell Physiol, 2015, 230(2): 318-326.
40. Chen J, Yang L, Guo L, et al. Sodium hyaluronate as a drug-release system for VEGF 165 improves graft revascularization in anterior cruciate ligament reconstruction in a rabbit model. Exp Ther Med, 2012, 4(3): 430-434.

Chinese Journal of Reparative and Reconstructive Surgery

In vitrodifferentiation of human amniotic mesenchymal stem cells into ligament fibroblasts after induced by transforming growth factor β₁ and vascular endothelial growth factor

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Chinese Journal of Reparative and Reconstructive Surgery

In vitrodifferentiation of human amniotic mesenchymal stem cells into ligament fibroblasts after induced by transforming growth factor β1 and vascular endothelial growth factor

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In vitrodifferentiation of human amniotic mesenchymal stem cells into ligament fibroblasts after induced by transforming growth factor β₁ and vascular endothelial growth factor