Scleraxis combined with basic fibroblast growth factor promotes the differentiation of human amniotic mesenchymal stem cells into human ligament fibroblasts in vitro

doi:10.3969/j.issn.2095-4344.1803

Abstract

Abstract:

BACKGROUND: Human amniotic mesenchymal stem cells have multi-directional differentiation ability. Studies have shown that both Scleraxis and basic fibroblast growth factor can promote the differentiation of human amniotic mesenchymal stem cells into human ligament fibroblasts.
OBJECTIVE: To explore whether Scleraxis combined with basic fibroblast growth factor can promote the differentiation of human amniotic mesenchymal stem cells into human ligament fibroblasts and to observe their differentiation effects.
METHODS: The study protocol was approved by the ethic committee of the Affiliated Hospital of Zunyi Medical University, and written informed consent was obtained from each puerpera. The amniotic membrane from the full-term placenta was separated, and human amniotic mesenchymal stem cells were isolated by a two-step enzyme digestion. The morphology of the cells was observed by inverted phase contrast microscope. The structure of fresh human amniotic membrane was observed by hematoxylin-eosin staining. The third generation of human amniotic mesenchymal stem cells were cultured in four groups: (1) normally cultured human amnion mesenchymal stem cell culture (simple culture group); (2) human amniotic mesenchymal stem cells infected with Scleraxis gene lentivirus (Scleraxis group); (3) human amniotic mesenchymal stem cells induced by basic fibroblast growth factor (basic fibroblast growth factor group); (4) human amniotic mesenchymal stem cells induced by Scleraxis and basic fibroblast growth factor (combined induction group). The proliferation ability of the cells in each group was detected by cell counting kit-8 method at 3 days of cell culture. Real-time quantitative PCR was performed to evaluate the cell differentiation into human ligament fibroblasts in each group at 14 days of culture.
RESULTS AND CONCLUSION: (1) Under the inverted phase contrast microscope, the third-generation human amniotic mesenchymal stem cells were long-fusiform and exhibited vortex-like adherent growth. (2) Under the inverted phase contrast microscope, the fresh human amniotic membrane had an obvious layered structure, which was divided into five layers: epithelial layer, basal layer, dense layer, fibroblast layer and sponge layer. (3) Human amniotic mesenchymal stem cells strongly and stably expressed green fluorescence at 24 hours after infection with Scleraxis gene lentivirus. The morphology of human amniotic mesenchymal stem cells induced by basic fibroblast growth factor was changed, and after 14 days of induction its morphology was similar to that of human ligament fibroblasts. (4) The results of cell counting kit-8 showed that the four groups of cells showed a S-type growth. The Scleraxis group, basic fibroblast growth factor group, combined induction group had stronger proliferation ability than the simple culture group, but there was no significant difference in the proliferative capacity between the three groups. (5) Real-time fluorescent quantitative PCR showed that the mRNA expression levels of ligament-related genes type I collagen, type III collagen, Fibronectin, Tenascin-C and TNMD in the Scleraxis group, basic fibroblast growth factor group, and combined induction group were significantly higher than those in the simple culture group (P < 0.05). The mRNA expression levels of ligament-related genes in the combined induction group were significantly higher than those in the Slckleris group and basic fibroblast growth factor group (P < 0.05). These results indicate that the combination of Scleraxis gene and basic fibroblast growth factor can promote the differentiation of human amniotic mesenchymal stem cells into human ligament fibroblasts, providing a new idea for the treatment of ligament injury.

Key words: human amniotic mesenchymal stem cells, Scleraxis, basic fibroblast growth factor, directed differentiation, human ligament fibroblasts, lentivirus infection, tissue engineering

CLC Number:

Sang Peng, Liu Yi. Scleraxis combined with basic fibroblast growth factor promotes the differentiation of human amniotic mesenchymal stem cells into human ligament fibroblasts in vitro[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(29): 4644-4650.

Figures/Tables 2

2.1 人羊膜间充质干细胞的形态倒置相差显微镜显示：40倍镜下第3代人羊膜间充质干细胞呈长梭形贴壁生长，可见漩涡形成，见图1A；100倍镜下可见细胞接触紧密，类似于三角形或长菱形，见图1B；200倍镜下观察细胞长梭状明显，细胞间相互黏附，见图1C。 2.2 倒置显微镜下羊膜的结构人新鲜羊膜经过切片、苏木精-伊红染色后在倒置显微镜下观察呈现明显的分层结构，一共分为5层，分别为上皮层、基底层、致密层、纤维母细胞层和海绵层，各层均比较明显且很完整，见图2。 2.3 慢病毒包装载体的构建、包装与滴度检测结果病毒梯度法感染293T细胞96 h后，荧光显微镜观察目的荧光表达量：0.03 μL/孔表达量约50%，0.10 μL/孔表达量约80%，0.30 μL/孔表达量约95%，见图3。 2.4 病毒感染人羊膜间充质干细胞 6.0 μL病毒原液感染人羊膜间充质干细胞96 h后荧光显示感染率良好，见图4。 2.5 碱性成纤维细胞生长因子诱导人羊膜间充质干细胞经过碱性成纤维细胞生长因子诱导的人羊膜间充质干细胞形态发生改变，14 d时最明显，细胞呈涡旋状，形似人韧带成纤维细胞，见图5。 2.6 CCK-8检测人羊膜间充质干细胞活性各组细胞的增殖能力均呈S型生长，其中Scleraxis基因慢病毒感染组、碱性成纤维细胞生长因子诱导组、联合诱导组较对照组增殖能力强(P < 0.05)，但这3组间的增殖能力差异无显著性意义(P > 0.05)，见图6，说明Scleraxis、碱性成纤维细胞生长因子不仅能够促进羊膜间充质干细胞分化，而且还能够促进其增殖。 2.7 实时荧光定量PCR检测慢病毒感染人羊膜间充质干细胞后Scleraxis的表达及韧带成纤维细胞相关基因的表达 Scleraxis基因慢病毒能够成功感染人羊膜间充质干细胞，感染后Scleraxis mRNA的表达量急剧升高，是未转染组的40倍左右，差异有显著性意义(P < 0.05)；Scleraxis基因慢病毒感染组、碱性成纤维细胞生长因子诱导组、联合诱导组韧带成纤维细胞相关基因mRNA的表达量均高于单纯人羊膜间充质干细胞培养组，差异有显著性意义(P < 0.05)，联合诱导组韧带相关基因的mRNA表达量高于Scleraxis基因慢病毒感染组和碱性成纤维细胞生长因子诱导组，差异有显著性意义(P < 0.05)，Scleraxis基因慢病毒感染组、碱性成纤维细胞生长因子诱导组韧带相关基因的表达差异无显著性意义(P > 0.05)，见图7。"

References

[1]Hinck AP, Mueller TD, Springer TA.Structural Biology and Evolution of the TGF-β Family. Cold Spring Harb Perspect Biol. 2016; 8(12): a022103.

[2]Iliadis DP, Bourlos DN, Mastrokalos DS, et al. LARS Artificial Ligament Versus ABC Purely Polyester Ligament for Anterior Cruciate Ligament Reconstruction. Orthop J Sports Med. 2016;4(6):2325967116653359.

[3]Spragg L, Chen J, Mirzayan R, et al. The Effect of Autologous Hamstring Graft Diameter on the Likelihood for Revision of Anterior Cruciate Ligament Reconstruction. Am J Sports Med. 2016;44(6):1475-1481.

[4]Thompson SM, Salmon LJ, Waller A, et al. Twenty-Year Outcome of a Longitudinal Prospective Evaluation of Isolated Endoscopic Anterior Cruciate Ligament Reconstruction With Patellar Tendon or Hamstring Autograft. Am J Sports Med. 2016;44(12):3083-3094.

[5]Proffen BL, Sieker JT, Murray MM. Bio-enhanced repair of the anterior cruciate ligament. Arthroscopy. 2015;31(5):990-997.

[6]Powers CJ, McLeskey SW, Wellstein A. Fibroblast growth factors, their receptors and signaling. Endocr Relat Cancer. 2000;7(3):165-197.

[7]Brancaleoni GH, Lourenzoni MR, Degrève L. Study of the influence of ethanol on basic fibroblast growth factor structure. Genet Mol Res. 2006;5(2):350-372.

[8]Ide J, Kikukawa K, Hirose J, et al. The effect of a local application of fibroblast growth factor-2 on tendon-to-bone remodeling in rats with acute injury and repair of the supraspinatus tendon. J Shoulder Elbow Surg. 2009;18(3):391-398.

[9]Yamamoto T, Wakitani S, Imoto K, et al. Fibroblast growth factor-2 promotes the repair of partial thickness defects of articular cartilage in immature rabbits but not in mature rabbits. Osteoarthritis Cartilage. 2004;12(8):636-641.

[10]Chuma H, Mizuta H, Kudo S, et al. One day exposure to FGF-2 was sufficient for the regenerative repair of full-thickness defects of articular cartilage in rabbits. Osteoarthritis Cartilage. 2004;12(10):834-842.

[11]Li X, Su G, Wang J, et al. Exogenous bFGF promotes articular cartilage repair via up-regulation of multiple growth factors. Osteoarthritis Cartilage. 2013;21(10):1567-1575.

[12]Wang H, Zou Q, Boerman OC, et al. Combined delivery of BMP-2 and bFGF from nanostructured colloidal gelatin gels and its effect on bone regeneration in vivo. J Control Release. 2013;166(2):172-181.

[13]Li Y, Liu Z, Jin Y, et al. Differentiation of Human Amniotic Mesenchymal Stem Cells into Human Anterior Cruciate Ligament Fibroblast Cells by In Vitro Coculture. Biomed Res Int. 2017;2017:7360354.

[14]Carlberg AL, Tuan RS, Hall DJ. Regulation of scleraxis function by interaction with the bHLH protein E47. Mol Cell Biol Res Commun. 2000;3(2):82-86.

[15]Hu JS, Olson EN, Kingston RE. HEB, a helix-loop-helix protein related to E2A and ITF2 that can modulate the DNA-binding ability of myogenic regulatory factors. Mol Cell Biol. 1992;12(3):1031-1042.

[16]Liu Y, Watanabe H, Nifuji A, et al. Overexpression of a single helix-loop-helix-type transcription factor, scleraxis, enhances aggrecan gene expression in osteoblastic osteosarcoma ROS17/2.8 cells. J Biol Chem. 1997;272(47):29880-29885.

[17]Alberton P, Popov C, Prägert M, et al. Conversion of human bone marrow-derived mesenchymal stem cells into tendon progenitor cells by ectopic expression of scleraxis. Stem Cells Dev. 2012;21(6):846-858.

[18]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[J]. 中国修复重建外科杂志, 2016,30(2):237-244.

[19]Filioli Uranio M, Dell'Aquila ME, Caira M, et al. Characterization and in 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.

[20]丁志,杨松林.间充质干细胞生物学特性及其分化潜能[J].中国组织工程研究与临床康复,2011,15(1):147-150.

[21]周文然,李新,王文波,等.人羊膜间充质干细胞生物学特征及向多巴胺能神经元样细胞的分化[J]. 中国组织工程研究, 2014,18(23):3682-3690.

[22]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.

[23]Nogami M, Kimura T, Seki S, et al. A Human Amnion-Derived Extracellular Matrix-Coated Cell-Free Scaffold for Cartilage Repair: In Vitro and In Vivo Studies. Tissue Eng Part A. 2016;22(7-8):680-688.

[24]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[J].中国实验血液学杂志, 2016,24(3):858-864.

[25]Hong Y. ACL injury: incidences, healing, rehabilitation, and prevention: part of the Routledge Olympic special issue collection. Res Sports Med. 2012;20(3-4):155-156.

[26]Zaffagnini S, Signorelli C, Bonanzinga T, et al. Technical variables of ACL surgical reconstruction: effect on post-operative static laxity and clinical implication. Knee Surg Sports Traumatol Arthrosc. 2016;24(11): 3496-3506.

[27]Ahn JH, Lee YS, Ko TS, et al. Accuracy and Reproducibility of the Femoral Tunnel With Different Viewing Techniques in the ACL Reconstruction. Orthopedics. 2016;39(6):e1085-e1091.

[28]Schweitzer R, Chyung JH, Murtaugh LC, et al. Analysis of the tendon cell fate using Scleraxis, a specific marker for tendons and ligaments. Development. 2001;128(19):3855-3866.

[29]Cserjesi P, Brown D, Ligon KL, et al. Scleraxis: a basic helix-loop-helix protein that prefigures skeletal formation during mouse embryogenesis. Development. 1995;121(4):1099-1110.

[30]Pryce BA, Brent AE, Murchison ND, et al. Generation of transgenic tendon reporters, ScxGFP and ScxAP, using regulatory elements of the scleraxis gene. Dev Dyn. 2007;236(6):1677-1682.

[31]Mendias CL, Gumucio JP, Bakhurin KI, et al. Physiological loading of tendons induces scleraxis expression in epitenon fibroblasts. J Orthop Res. 2012;30(4):606-612.

[32]Murchison ND, Price BA, Conner DA, et al. Regulation of tendon differentiation by scleraxis distinguishes force-transmitting tendons from muscle-anchoring tendons. Development. 2007;134(14):2697-2708.

[33]Alberton P, Popov C, Prägert M, et al. Conversion of human bone marrow-derived mesenchymal stem cells into tendon progenitor cells by ectopic expression of scleraxis. Stem Cells Dev. 2012;21(6):846-858.

[34]Gulotta LV, Rodeo SA. Emerging ideas: Evaluation of stem cells genetically modified with scleraxis to improve rotator cuff healing. Clin Orthop Relat Res. 2011;469(10):2977-2980.

[35]Gulotta LV, Kovacevic D, Packer JD, et al. Bone marrow-derived mesenchymal stem cells transduced with scleraxis improve rotator cuff healing in a rat model. Am J Sports Med. 2011;39(6):1282-1289.

[36]Longo UG, Lamberti A, Maffulli N, et al. Tissue engineered biological augmentation for tendon healing: a systematic review. Br Med Bull. 2011;98:31-59.

[37]Durgam SS, Stewart AA, Pondenis HC, et al. Responses of equine tendon- and bone marrow-derived cells to monolayer expansion with fibroblast growth factor-2 and sequential culture with pulverized tendon and insulin-like growth factor-I. Am J Vet Res. 2012;73(1):162-170.

[38]Brent AE, Tabin CJ. FGF acts directly on the somitic tendon progenitors through the Ets transcription factors Pea3 and Erm to regulate scleraxis expression. Development. 2004;131(16):3885-3896.

[39]陈志达,蔡弢艺,吴进,等. 成纤维细胞生长因子诱导骨髓间充质干细胞分化腱细胞的研究[J]. 中国骨与关节损伤杂志, 2017,32(8):819-822.

[40]Chen CH, Cao Y, Wu YF, et al. Tendon healing in vivo: gene expression and production of multiple growth factors in early tendon healing period. J Hand Surg Am. 2008;33(10):1834-1842.

[41]Thomopoulos S, Das R, Sakiyama-Elbert S, et al. bFGF and PDGF-BB for tendon repair: controlled release and biologic activity by tendon fibroblasts in vitro. Ann Biomed Eng. 2010;38(2):225-234.