一种新型声带生物反应器的构建与鉴定
Summary
A novel vocal fold bioreactor capable of delivering physiologically relevant, vibratory stimulation to cultured cells is constructed and characterized. This dynamic culture device, when combined with a fibrous poly(ε-caprolactone) scaffold, creates a vocal fold-mimetic environment that modulates the behaviors of mesenchymal stem cells.
Abstract
In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To emulate the dynamic environment of vocal folds, a novel vocal fold bioreactor capable of producing vibratory stimulations at fundamental phonation frequencies is constructed and characterized. The device is composed of a function generator, a power amplifier, a speaker selector and parallel vibration chambers. Individual vibration chambers are created by sandwiching a custom-made silicone membrane between a pair of acrylic blocks. The silicone membrane not only serves as the bottom of the chamber but also provides a mechanism for securing the cell-laden scaffold. Vibration signals, generated by a speaker mounted underneath the bottom acrylic block, are transmitted to the membrane aerodynamically by the oscillating air. Eight identical vibration modules, fixed on two stationary metal bars, are housed in an anti-humidity chamber for long-term operation in a cell culture incubator. The vibration characteristics of the vocal fold bioreactor are analyzed non-destructively using a Laser Doppler Vibrometer (LDV). The utility of the dynamic culture device is demonstrated by culturing cellular constructs in the presence of 200-Hz sinusoidal vibrations with a mid-membrane displacement of 40 µm. Mesenchymal stem cells cultured in the bioreactor respond to the vibratory signals by altering the synthesis and degradation of vocal fold-relevant, extracellular matrix components. The novel bioreactor system presented herein offers an excellent in vitro platform for studying vibration-induced mechanotransduction and for the engineering of functional vocal fold tissues.
Introduction
人类的声带，上皮层构成，固有层（LP）和声带肌肉，是一种将来自肺部的气流进入的声波产生声音的专门的软组织。1声带定期正常发声期间振荡，表现出高达30％的菌株在基本频率范围从100至300赫兹。2成人声带唱片是肤浅（SLP）组成的梯度结构，中间（ILP）和深（DLP）层。进一步分类组的上皮和SLP为粘膜层，并结合ILP和DLP到声韧带。3 SLP层主要包含一种无定形矩阵稀疏分散的胶原纤维，而韧带富含成熟的胶原蛋白和弹性纤维提供足够的强度。4的结构和新生儿声带力学从他们成熟的同行有所不同显著。虽然机制•调控声带发育和成熟都还没有完全理解，实验证据指出，以发声来源的机械应力的决定性作用。
几个医疗条件，包括滥用嗓音，感染，化学刺激和手术过程中，可能会损坏声带。声带疾病影响美国人口估计有3-9％。目前的治疗方法声带疾病是有限的5和基于干细胞的组织工程方法已经成为一种很有前途的战略恢复声带功能。间充质干细胞（MSCs）是一个合适的替代品主要声带成纤维细胞的声带组织工程。6-9干细胞的命运规范和随后的组织发展是由它们驻留在特定的利基，其中的机械条件是介个至关重要的因素。10机械力的组织形态发生一个重要的监管机构第二平衡，特别是对于那些定期进行装载11从组织工程的角度来看的组织，它已被证明，暴露于生理相关的机械刺激促进干细胞分化和组织特异性基质重塑。12-15
组织培养物的生物反应器被设计来模拟所需的生理环境，细胞或组织生长的体外 。对声带组织工程，这是特别重要的，以重新创建phonating声带的机械环境。一个理想的声带生物反应器应有效地提供振动提示来培养细胞，让轻便的控制频率，振幅和振动持续时间。 Titze和同事设计了一种声带生物反应器（T1生物反应器 ）16，结合了静态拉伸与高频（20-200赫兹）振动来刺激基质蛋白的细胞产量。使用设置克该生物反应器，Webb和同事研究了17的10天，100赫兹的振动对皮肤成纤维细胞中透明质酸（HA）为基础的水凝胶培养的影响。构建体进行振动表现出升高的表达HA合酶-2（HAS2），核心蛋白聚糖，纤维调节和基质金属蛋白酶1（MMP1），相对于静态控件。被发现的刺激作用是时间依赖性的。最近，使用了功率放大器，一个函数发生器，一个封闭的扬声器和一个沿圆周锚硅油膜，该振荡空气传送到贴壁细胞我们组18组装声带生物反应器（J1生物反应器 ）。培养在生物反应器中的J1新生儿包皮成纤维细胞，进行1小时的振动，在60，110或300赫兹，带有一个在平面应变高达0.05％。的定量PCR结果显示，一些细胞外基质基因的表达被适当地改变以响应变化的振动频率和幅度。
这些生物反应器的设计，而耐人寻味，有一些局限性。例如，在T1的系统需要大量的连接器和棒的机械耦合的，从而限制了最大频率达到的。此外，细胞可以经受不期望的机械搅拌和流体扰动变得复杂的数据解释。 J1的生物反应器，在另一方面，具有相对较低的能量转换效率，而不是用户友好。此外，频繁的振动分离从底层硅油膜细胞载货结构。这里报道的J2声带生物反应器，基于相同的原理为J1版本设计的，被优化的一致性和可重复性。在空气动力学中单独装配的振动室，其中的MSC-纤维状填充的聚（ε-己内酯）（PCL）支架是effectiv产生的发声-模拟振动伊利抵押。激光多普勒Vibrometry（LDV）允许用户以验证膜/支架组件的振动模式。在我们的演示中，干细胞暴露在200 Hz的正弦振动与1小时的按1小时 – 解除（OF）模式，共12小时，每天的7天。细胞反应所施加的振动线索进行了系统研究。总体而言，J2声带提供了最人性化的功能，允许动态细胞培养的研究，高通量和可重复的方式进行。
Protocol
Representative Results
Discussion
功能性声带组织在体外成功的工程需要声带般的微环境介导多能细胞的行为的娱乐。人们普遍认为组织或器官的结构反映他们需要执行的功能。对于22声带组织，提出发声过程中发生的高频振动来对组织成熟的重要。在我们的研究中，PCL支架是用来提供一个带样结构支撑而声带生物反应器被设计成引入生理学相关的机械信号到培养的干细胞。这里描述的（J2）的生物反应器，?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢杰弗里·卡普兰博士为他的共焦成像的培训和咨询。我们也感谢凯克电子显微镜实验室和超英博士倪扫描电镜的协助。这项工作是由卫生（NIDCD，R01DC008965和R01DC011377）国家研究院资助。 ABZ承认美国国家科学基金会综合研究生教育和研究培训实习资助（IGERT）计划。
Materials
| silicone elastomer kit | Dow Corning | Sylgard 184 | cure the membrane at 100 C for 2 hr |
| PCL | Sigma Aldrich | 440744-500G | Mn ~ 80 kDa, dissolve overnight |
| chloroform | Sigma Aldrich | C7559-5VL | |
| human bone marrow-derived MSCs | Lonza | PT-2501 | received with passage 2 |
| MSC maintenance media | Lonza | PT-3001 | 10% FBS in basal media supplemented with L-glutamine, gentamicin and amphotericin |
| Accutase cell dissociation reagent | Life Technologies | A11105-01 | |
| ethanol | Sigma Aldrich | E7023-500ML | |
| fibronectin | Sigma Aldrich | F2006-1MG | |
| MMP1 DuoSet ELISA kit | R&D systems | DY901 | |
| HA ELISA kit | Echelon Biosciences | K-1200 | |
| PBS | Life Technologies | 14190-136 | |
| propidium iodide | Life Technologies | P1304MP | |
| Syto-13 | Life Technologies | S7575 | |
| QuantiTect reverse transcription kit | Qiagen | 205311 | |
| SYBR Green PCR master mix | Life Technologies | 4309155 | |
| replacement speaker | DAYTON audio (via Parts Express) |
DS90-8 | paper cone, full range (80-13000 Hz), 85dB |
| Ergo Micro torque screwdriver | Mountz | # 020377 | torque range: 20-120 cN.m |
| stereo speaker selector | RadioShack | 40-244 | maximum power handling 50 W |
| function generator | Agilent | 33220A | frequency range 1 µHz- 20 MHz |
| power amplifier | PYLE audio | PylePro PT2400 | frequency response: 10 Hz-50 kHz, two speaker channels |
| cell culture incubator | Thermo Fisher | Steri-Cult 3307 | |
| syringe pump | New Era Pump Systems | NE-300 | |
| High voltage power supply | Spellman | CZE 1000R | output voltage: 0-30 kV |
| scanning electron microscope | JEOL-USA | JSM-7400F | |
| desk gold sputter coater | Denton Vacuum | DSK00V-0013 | |
| Doppler laser vibrometer | Polytec | PDV-100 | non-contact velocity measurement (0-22 kHz) |
| PCR sequence detection system | Applied Biosystems | ABI7300 | |
| multiphoton confocal microscope | Zeiss | Zeiss 510Meta NLO | |
| UV-VIS Spectrophotometer | NanoDrop Products via Thermo Scientific |
ND-2000 | |
| VibSoft Data Acquisition Software | Polytec | acquisition bandwidth up to 40 MHz | |
| Origin 8.5 data analysis software | OriginLab | ||
| qbasePlus qPCR data analysis software | Biogazelle | V2.3 | |
| aluminium alloy | McMaster-Carr | Alloy 6061 | |
| acrylic blocks | McMaster-Carr | ||
| polycarbonate anti-humidity chamber | McMaster-Carr | Impact-Resistant Polycarbonate | |
| screws | McMaster-Carr | ||
| electronic cable/wire | |||
| medical grade PVC tubing | US Plastic Corp. | Tygon S-50-HL | clear, biocompatible |
| 10 mL syringe | Becton Dickinson | 309604 | |
| 21 G blunt ended needle | Small Parts | NE-213PL-25 | 1-1/2" length |
| Alligator clip adapters | RadioShack | 270-354 | fully insulated |
| 8 mm biopsy punch | Sklar Surgical Instruments | 96-1152 | sterile, disposable |
| 12 mm biopsy punch | Acuderm (via Fisher Scientific) | NC9998681 | |
| tissue culture flasks | Corning | cell culture treated |
References
- Titze, I. R. Mechanical-Stress in Phonation. J. Voice. 8, 99-105 (1994).
- Titze, I. R. On the Relation Between Subglottal Pressure and Fundamental-Frequency in Phonation. J. Acoust. Soc. Am. 85, 901-906 (1989).
- Gray, S. D. Cellular physiology of the vocal folds. Otolaryngol. Clin. N. Am. 33, 679-698 (2000).
- Thibeault, S. L., Gray, S. D., Bless, D. M., Chan, R. W., Ford, C. N. Histologic and rheologic characterization of vocal fold scarring. J. Voice. 16, 96-104 (2002).
- Hansen, J. K., Thibeault, S. L. Current understanding and review of the literature: Vocal fold scarring. J. Voice. 20, 110-120 (2006).
- Pittenger, M. F., et al. Multilineage potential of adult human mesenchymal stem cells. Science. 284, 143-147 (1999).
- Hanson, S. E., et al. Characterization of Mesenchymal Stem Cells From Human Vocal Fold Fibroblasts. Laryngoscope. 120, 546-551 (2010).
- Tong, Z., Duncan, R. L., Jia, X. Modulating the behaviors of mesenchymal stem cells via the combination of high-frequency vibratory stimulations and fibrous scaffolds. Tissue Eng. Part A. 19, 1862-1878 (2013).
- Tong, Z., Sant, S., Khademhosseini, A., Jia, X. Controlling the Fibroblastic Differentiation of Mesenchymal Stem Cells Via the Combination of Fibrous Scaffolds and Connective Tissue Growth Factor. Tissue Eng. Part A. 17, 2773-2785 (2011).
- Jones, D. L., Wagers, A. J. No place like home: anatomy and function of the stem cell niche. Nat. Rev. Mol. Cell Biol. 9, 11-21 (1038).
- Wang, J. H., Thampatty, B. P. Mechanobiology of Adult and Stem Cells. Int. Rev. of Cell Mol. Biol. 271, 301-346 (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 Eng. Part A. 16, 3457-3466 (2010).
- Kim, B. S., Nikolovski, J., Bonadio, J., Mooney, D. J. Cyclic mechanical strain regulates the development of engineered smooth muscle tissue. Nat. Biotechnol. 17, 979-983 (1999).
- Webb, K., et al. Cyclic strain increases fibroblast proliferation, matrix accumulation, and elastic modulus of fibroblast-seeded polyurethane constructs. J. Biomech. 39, 1136-1144 (2006).
- Kim, Y. J., Sah, R. L. Y., Grodzinsky, A. J., Plaas, A. H. K., Sandy, J. D. Mechanical Regulation of Cartilage Biosynthetic Behavior – Physical Stimuli.. Arch. Biochem. Biophys. 311, 1-12 (1994).
- Titze, I. R., et al. Design and validation of a bioreactor for engineering vocal fold tissues under combined tensile and vibrational stresses. J. Biomech. 37, 1521-1529 (2004).
- Kutty, J. K., Webb, K. Vibration stimulates vocal mucosa-like matrix expression by hydrogel-encapsulated fibroblasts. J. Tissue Eng. Regen. Med. 4, 62-72 (2010).
- Farran, A. J. E., et al. Design and Characterization of a Dynamic Vibrational Culture System. J. Tissue Eng. Regen. Med. , (2011).
- Reneker, D. H., Yarin, A. L. Electrospinning jets and polymer nanofibers. Polymer. 49, 2387-2425 (2008).
- Wang, Y., Theobald, P., Tyrer, J., Lepper, P. The application of scanning vibrometer in mapping ultrasound fields. J. Phys.: Conf. Ser. 1, 167-173 (2004).
- Brown, W. S., Morris, R. J., Hollien, H., Howell, E. Speaking Fundamental-Frequency Characteristics as a Function of Age and Professional. J. Voice. 5, 310-315 (1991).
- Ingber, D. E. Cellular mechanotransduction: putting all the pieces together again. Faseb J. 20, 811-827 (2006).
- Titze, I. R., et al. Design and validation of a bioreactor for engineering vocal fold tissues under combined tensile and vibrational stresses. J. Biomech. 37, 1521-1529 (2004).
- Moore, J., Thibeault, S. Insights Into the Role of Elastin in Vocal Fold Health and Disease. J. Voice. 26, 269-275 (2012).
- Thibeault, S. L., Bless, D. M., Gray, S. D. Interstitial protein alterations in rabbit vocal fold with scar. J. Voice. 17, 377-383 (2003).
- Branski, R. C., Verdolini, K., Sandulache, V., Rosen, C. A., Hebda, P. A. Vocal fold wound healing: A review for clinicians. J. Voice. 20, 432-442 (2006).
- Clark, I. A., Swingler, T. E., Sampieri, C. L., Edwards, D. R. The regulation of matrix metalloproteinases and their inhibitors. Int. J. Biochem. Cell B. 40, 1362-1378 (2008).
- Silver, F. H., Horvath, I., Foran, D. J. Viscoelasticity of the vessel wall: The role of collagen and elastic fibers. Crit. Rev. Biomed. Eng. 29, 279-301 (2001).
- Kutty, J. K., Webb, K. Tissue Engineering Therapies for the Vocal Fold Lamina Propria. Tissue Eng. Part B: Rev. 15, 249-262 (2009).
