应用三维单轴机械刺激生物反应器系统诱导肌生衍生干细胞的畸因分化
Summary
三维单轴机械刺激生物反应器系统是肌腱衍生干细胞和新肌腱形成具有致源特异性分化的理想生物反应器。
Abstract
肌腱病是一种常见的慢性肌腱疾病，与骨科区域的炎症和退化有关。由于发病率高，自我修复能力有限，最重要的是，没有明确的治疗，肌腱病仍然对患者的生活质量产生负面影响。肌腱衍生干细胞（TDSCs）作为肌腱细胞的原发性前体细胞，在肌腱病的发育以及肌腱病后的功能和结构恢复都起着至关重要的作用。因此，一种在体外模拟TDSC在体内分化为肌腱细胞的方法将是有用的。在这里，本协议描述了一种基于三维（3D）单轴拉伸系统的方法，以刺激TDSC分化成跟腱样组织。本方案分为七个阶段：小鼠TDSC的分离、小鼠TDSC的培养和扩张、细胞表形成刺激培养的制备、刺激培养的细胞片的形成、3D肌腱干细胞结构的准备、单轴拉伸机械刺激复合物的组装以及体外肌腱类组织的机械刺激评估。组织学证明了这种有效性。整个过程需要不到3周的时间。为了促进细胞外基质沉积，在刺激培养基中使用了4.4mg/mL抗坏血酸。带线性电机的分离室提供精确的机械载荷，便于携带且易于调整，适用于生物反应器。本协议的装载方案为6%应变，0.25赫兹，8小时，随后16小时休息6天。该协议可以模拟肌腱的细胞分化，有助于肌腱病的病理过程研究。此外，肌腱般的组织有可能用于促进肌腱愈合在肌腱损伤作为工程自体移植。综上关于本议定书简单、经济、可重复、有效的方案。
Introduction
肌腱病是常见的运动损伤之一。它主要表现在疼痛、局部肿胀、受影响区域肌肉张力减轻和功能障碍。肌腱病的发生率很高。跟腱病的存在是最常见的中长跑运动员（高达29%），而在排球运动员（45%），篮球（32%），田径（23），手球（15%）和足球（13%）1，2，3，4，51,2,3,4的运动员也高。然而，由于肌腱自我修复能力有限，缺乏有效的治疗方法，肌腱病仍然对患者的生活产生负面影响,。此外，肌腱病的发病机制尚不清楚。关于其发病机制的研究已经很多，主要包括”炎症论”，”退化论”，”过度使用理论“等。目前，许多研究人员认为，肌腱病是由于自我修复失败造成的微损伤造成的过度机械负荷肌腱经验9,9，10。
肌腱衍生干细胞（TDSCs）作为肌腱细胞的原发性前体细胞，在肌腱病,11、12、13,12之后，在肌腱病的发育和功能及结构恢复中起着至关重要的作用。据报道，机械应激刺激可引起骨细胞、成骨细胞、平滑肌细胞、成纤维细胞、中质干细胞和其他力敏感细胞的增殖和分化，14、15、16、17、18。14,15,16,17,18因此，TDSC，作为机械敏和多能细胞之一，同样可以通过机械载荷19，20来刺激，以区分。
然而，不同的机械载荷参数（装载强度、装载频率、载荷类型和装载周期）可以诱导TDSC分化成不同的单元21。因此，一个有效和有效的机械载荷制度对于肿瘤的发生具有非常重要意义。此外，还有不同类型的生物反应器作为刺激系统，目前用于向TDSC提供机械载荷。每种生物反应器的原理不同，因此对应不同生物反应器的机械载荷参数也不同。因此，需要一种简单、经济、可重复的刺激方案，包括生物反应器的类型、相应的刺激介质和机械载荷装置。
本文介绍了一种基于三维（3D）单轴拉伸系统的方法，以刺激TDSC分化成肌腱样组织。该协议分为七个阶段：小鼠TDSC的分离、小鼠TDSC的培养和扩张、细胞表形成刺激培养的制备、刺激培养的细胞表的形成、3D肌腱干细胞结构的准备、单轴拉伸机械刺激复合物的组装以及机外肌腱类组织的评价。整个过程需要不到3周的时间才能获得3D细胞构造，这远远低于一些现有的方法22，23。22,本协议已被证明能够诱导TDSC分化成肌腱组织，它比目前常用的二维（2D）拉伸系统21更可靠。组织学证明了这种有效性。简言之，本议定书简单、经济、可重复、有效。
Protocol
上述方法根据西澳大利亚大学动物伦理委员会的准则和条例得到批准和执行。 1. 小鼠TDSC的隔离 通过宫颈脱位使6-8周大的C57BL/6小鼠安乐死。 收获的跟腱24和跟腱25。 消化肌腱从一个与6 mL的一型我胶原酶（3毫克/mL）为3小时。注意：由于鼠标肌腱的大小较小，因此应在此步骤中使用从一只鼠标中采集的所有肌腱。</li…
Representative Results
在机械刺激之前，TDSC以完整的介质生长到100%的汇合，并表现出一种混乱的超结构形态（图2A）。经过6天的单轴拉伸机械载荷，细胞外矩阵（ECM）和细胞对齐方向良好（图2B）。机械装载后，电池填充良好，并密罩在 ECM 中。细胞形态呈现为拉长，与没有拉伸的肌腱细胞相比更相似（图2C）。带载荷的细胞构造中的细胞密度高于无载荷的细胞密度。QPCR结果表明?…
Discussion
肌腱是一种机械性纤维结缔组织。根据先前的研究，机械负荷过多可能导致肌腱干细胞的骨质分化，而负荷不足会导致肌腱分化期间胶原纤维结构紊乱。
一个普遍的观点是，理想的生物反应器的关键是能够模拟体内细胞微环境，细胞在体内经历的能力。因此，在体外模仿体内正常应激环境是刺激TDSC单谱分化到肌腱细胞的关键。在目前的协议中，TDSC构造的宽…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项研究是在提交人获得”西澳大利亚大学国际学费奖学金和西澳大利亚大学研究生奖”时进行的。这项工作得到了国家自然科学基金（81802214）的支持。
Materials
| Ascorbic acid | Sigma-aldrich | PHR1008-2G | |
| Fetal bovine serum (FBS) | Gibcoä by Life Technologies | 1908361 | |
| Histology processor | Leica | TP 1020 | |
| Minimal Essential Medium (Alpha-MEM) | Gibcoä by Life Technologies | 2003802 | |
| Mouse Tendon Derived Stem Cell | Isolated from Achilles tendons of 6- to 8-wk-old C57BL/6 mice. Then digested with type I collagenase (3 mg/ml; MilliporeSigma, Burlington, MA, USA) for 3 h and passed through a 70 mmcell strainer to yield single-cell suspensions. | ||
| Paraformaldehyde | Sigma-aldrich | 441244 | |
| Streptomycin and penicillin mixture | Gibcoä by Life Technologies | 15140122 | |
| Three-dimensional Uniaxial Mechanical Stimulation Bioreactor System | Centre of Orthopaedic Translational Research, Medical School, University of Western Australia | Available from the corresponding author upon request. Or make it according to our design* *Wang T, Lin Z, Day RE, et al. Programmable mechanical stimulation influences tendon homeostasis in a bioreactor system. Biotechnol Bioeng. 2013;110(5):1495–1507. doi:10.1002/bit.24809 | |
| Trypsin | Gibcoä by Life Technologies | 1858331 |
Riferimenti
- Knobloch, K., Yoon, U., Vogt, P. M. Acute and overuse injuries correlated to hours of training in master running athletes. Foot & Ankle International. 29 (7), 671-676 (2008).
- Kujala, U. M., Sarna, S., Kaprio, J. Cumulative incidence of achilles tendon rupture and tendinopathy in male former elite athletes. Clinical Journal of Sport Medicine. 15 (3), 133-135 (2005).
- Lian, O. B., Engebretsen, L., Bahr, R. Prevalence of jumper’s knee among elite athletes from different sports: a cross-sectional study. The American Journal of Sports Medicine. 33 (4), 561-567 (2005).
- Zwerver, J., Bredeweg, S. W., vanden Akker-Scheek, I. Prevalence of Jumper’s knee among nonelite athletes from different sports: a cross-sectional survey. The American Journal of Sports Medicine. 39 (9), 1984-1988 (2011).
- van der Worp, H., et al. Risk factors for patellar tendinopathy: a systematic review of the literature. British Journal of Sports Medicine. 45 (5), 446-452 (2011).
- Lopez, R. G. L., Jung, H. -. G. Achilles tendinosis: treatment options. Clinics in Orthopedic Surgery. 7 (1), 1-7 (2015).
- Wren, T. A., Yerby, S. A., Beaupré, G. S., Carter, D. R. Mechanical properties of the human achilles tendon. Clinical Biomechanics. 16 (3), 245-251 (2001).
- Rees, J. D., Wilson, A. M., Wolman, R. L. Current concepts in the management of tendon disorders. Rheumatology. 45 (5), 508-521 (2006).
- Magnan, B., Bondi, M., Pierantoni, S., Samaila, E. The pathogenesis of Achilles tendinopathy: a systematic review. Foot and Ankle Surgery. 20 (3), 154-159 (2014).
- Riley, G. The pathogenesis of tendinopathy. A molecular perspective. Rheumatology. 43 (2), 131-142 (2004).
- Bi, Y., et al. Identification of tendon stem/progenitor cells and the role of the extracellular matrix in their niche. Nature Medicine. 13 (10), 1219-1227 (2007).
- Zhang, J., Wang, J. H. C. BMP-2 mediates PGE(2) -induced reduction of proliferation and osteogenic differentiation of human tendon stem cells. Journal of Orthopaedic Research. 30 (2), 47-52 (2012).
- Chen, L., et al. Synergy of tendon stem cells and platelet-rich plasma in tendon healing. Journal of Orthopaedic Research. 30 (6), 991-997 (2012).
- Wang, J., et al. Mechanical stimulation orchestrates the osteogenic differentiation of human bone marrow stromal cells by regulating HDAC1. Cell Death & Disease. 7 (5), 2221 (2016).
- Parvizi, M., Bolhuis-Versteeg, L. A. M., Poot, A. A., Harmsen, M. C. Efficient generation of smooth muscle cells from adipose-derived stromal cells by 3D mechanical stimulation can substitute the use of growth factors in vascular Tissue Engineeringineering. Biotechnology Journal. 11 (7), 932-944 (2016).
- Sun, L., et al. Effects of Mechanical Stretch on Cell Proliferation and Matrix Formation of Mesenchymal Stem Cell and Anterior Cruciate Ligament Fibroblast. Stem Cells International. 2016, 9842075 (2016).
- Lin, X., Shi, Y., Cao, Y., Liu, W. Recent progress in stem cell differentiation directed by material and mechanical cues. Biomedical Materials. 11 (1), 014109 (2016).
- Li, R., et al. Mechanical strain regulates osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells. Biomed Research International. 2015, 873251 (2015).
- Zhang, J., Wang, J. H. C. Characterization of differential properties of rabbit tendon stem cells and tenocytes. BMC Musculoskeletal Disorders. 11, 10-10 (2010).
- Liu, X., Chen, W., Zhou, Y., Tang, K., Zhang, J. Mechanical Tension Promotes the Osteogenic Differentiation of Rat Tendon-derived Stem Cells Through the Wnt5a/Wnt5b/JNK Signaling Pathway. Cellular Physiology and Biochemistry. 36 (2), 517-530 (2015).
- Wang, T., et al. 3D uniaxial mechanical stimulation induces tenogenic differentiation of tendon-derived stem cells through a PI3K/AKT signaling pathway. FASEB Journal. 32 (9), 4804-4814 (2018).
- Calve, S., et al. Engineering of functional tendon. Tissue Engineering. 10 (5-6), 755-761 (2004).
- Kostrominova, T. Y., Calve, S., Arruda, E. M., Larkin, L. M. Ultrastructure of myotendinous junctions in tendon-skeletal muscle constructs engineered in vitro. Histology & Histopathology. 24 (5), 541-550 (2009).
- Wagner, J. R., Taguchi, T., Cho, J. Y., Charavaryamath, C., Griffon, D. J. Evaluation of Stem Cell Therapies in a Bilateral Patellar Tendon Injury Model in Rats. Journal of Visualized Experiments. (133), e56810 (2018).
- Kurtaliaj, I., Golman, M., Abraham, A. C., Thomopoulos, S. Biomechanical Testing of Murine Tendons. Journal of Visualized Experiments. (152), e60280 (2019).
- Hsiao, M. Y., et al. The Effect of the Repression of Oxidative Stress on Tenocyte Differentiation: A Preliminary Study of a Rat Cell Model Using a Novel Differential Tensile Strain Bioreactor. International Journal of Molecular Sciences. 20 (14), (2019).
- Morita, Y., et al. The optimal mechanical condition in stem cell-to-tenocyte differentiation determined with the homogeneous strain distributions and the cellular orientation control. Biology Open. 8 (5), 0339164 (2019).
- Shukunami, C., Oshima, Y., Hiraki, Y. Molecular cloning of tenomodulin, a novel chondromodulin-I related gene. Biochemical and Biophysical Research Communications. 280 (5), 1323-1327 (2001).
- Murchison, N. D., et al. Regulation of tendon differentiation by scleraxis distinguishes force-transmitting tendons from muscle-anchoring tendons. Development. 134 (14), 2697-2708 (2007).
- Liu, W., et al. The atypical homeodomain transcription factor Mohawk controls tendon morphogenesis. Molecular and Cellular Biology. 30 (20), 4797-4807 (2010).
- Chang, J., Thunder, R., Most, D., Longaker, M. T., Lineaweaver, W. C. Studies in flexor tendon wound healing: neutralizing antibody to TGF-beta1 increases postoperative range of motion. Plastic and Reconstructive Surgery. 105 (1), 148-155 (2000).
- Bennett, N. T., Schultz, G. S. Growth factors and wound healing: biochemical properties of growth factors and their receptors. American Journal of Surgery. 165 (6), 728-737 (1993).
- Wòjciak, B., Crossan, J. F. The effects of T cells and their products on in vitro healing of epitenon cell microwounds. Immunology. 83 (1), 93-98 (1994).
- Marui, T., et al. Effect of growth factors on matrix synthesis by ligament fibroblasts. Journal of Orthopaedic Research. 15 (1), 18-23 (1997).
- Ni, M., et al. Engineered scaffold-free tendon tissue produced by tendon-derived stem cells. Biomaterials. 34 (8), 2024-2037 (2013).
- Trumbull, A., Subramanian, G., Yildirim-Ayan, E. Mechanoresponsive musculoskeletal tissue differentiation of adipose-derived stem cells. Biomedical Engineering Online. 15, 43 (2016).
- Wang, T., et al. Programmable mechanical stimulation influences tendon homeostasis in a bioreactor system. Biotechnology and Bioengineering. 110 (5), 1495-1507 (2013).
- Nirmalanandhan, V. S., et al. Effect of scaffold material, construct length and mechanical stimulation on the in vitro stiffness of the engineered tendon construct. Journal of Biomechanics. 41 (4), 822-828 (2008).
- Doroski, D. M., Levenston, M. E., Temenoff, J. S. Cyclic tensile culture promotes fibroblastic differentiation of marrow stromal cells encapsulated in poly(ethylene glycol)-based hydrogels. Tissue Engineeringineering. Part A. 16 (11), 3457-3466 (2010).
- Altman, G. H., et al. Advanced bioreactor with controlled application of multi-dimensional strain for Tissue Engineeringineering. Journal of Biomechanical Engineering. 124 (6), 742-749 (2002).
- Webb, K., et al. Cyclic strain increases fibroblast proliferation, matrix accumulation, and elastic modulus of fibroblast-seeded polyurethane constructs. Journal of Biomechanics. 39 (6), 1136-1144 (2006).
- Parent, G., Huppé, N., Langelier, E. Low stress tendon fatigue is a relatively rapid process in the context of overuse injuries. Annals of Biomedical Engineering. 39 (5), 1535-1545 (2011).
- Wang, T., et al. Bioreactor design for tendon/ligament engineering. Tissue Engineeringineering. Part B, Reviews. 19 (2), 133-146 (2013).
- Smith, R. K. Mesenchymal stem cell therapy for equine tendinopathy. Disability and Rehabilitation. 30 (20-22), 1752-1758 (2008).
- Godwin, E. E., Young, N. J., Dudhia, J., Beamish, I. C., Smith, R. K. Implantation of bone marrow-derived mesenchymal stem cells demonstrates improved outcome in horses with overstrain injury of the superficial digital flexor tendon. Journal of Equine Veterinary Science. 44 (1), 25-32 (2012).
- Lacitignola, L., Crovace, A., Rossi, G., Francioso, E. Cell therapy for tendinitis, experimental and clinical report. Veterinary Research Communications. 32, 33-38 (2008).
- Del Bue, M., et al. Equine adipose-tissue derived mesenchymal stem cells and platelet concentrates: their association in vitro and in vivo. Veterinary Research Communications. 32, 51-55 (2008).
- Awad, H. A., et al. Repair of patellar tendon injuries using a cell-collagen composite. Journal of Orthopaedic Research. 21 (3), 420-431 (2003).
Citazione di questo articolo
Chen, Z., Chen, P., Ruan, R., Chen, L., Yuan, J., Wood, D., Wang, T., Zheng, M. H. Applying a Three-dimensional Uniaxial Mechanical Stimulation Bioreactor System to Induce Tenogenic Differentiation of Tendon-Derived Stem Cells. J. Vis. Exp. (162), e61278, doi:10.3791/61278 (2020).