Tissue Engineering Bioreactor by: Karen Chen, Richard Bamberg, Rachel Mosher, Dustin Gardner Advisor: Dr. Brenda Ogle Client: Dr. Susan Thibeault Date: March 14, 2007 1Table of Contents Abstract (pg. 2) Background (pg. 2-8) Alternative Design Descriptions (pg. 8-11) Final Design (pg. 11-13) References (pg. 13) Appendix A: Product Design Specifications (pg. 13-16) Abstract:In the interest of pursuing further research into vocal fold cells, a bioreactor was constructed to effectively simulate the environment of the vocal fold tissue within the human body. The purpose of the design was to subject cell-seeded strips (vocal fold substitutes) to a variety of vibration and tensile stimulation so that vocal fold cells may be studied more in-depth. Only one such bioreactor is known to exist, however the objective for this design project is to reconstruct a similar bioreactor and make a few modifications to the device that would enhance its performance for research. For this project, the single-side vibration bar with parallel strips will be constructed due to its high score in the design matrix. Background: Bioreactor: A bioreactor is a system or device that supports a biological system. The purpose of a bioreactor is to simulate an active environment that is close to a biological system. In this system, organisms or tissues of organisms are applied with known variables, and the results due to the variables would be observed. This system has various sizes, could be as large as cubic meters and as small as a Petri dish. The material of the bioreactor could be stainless steel or polyvinylchloride (PVC). 2In this case, an environment that is very close to the larynx will be simulated. A T-75 culture flask made of PVC will be used as the bioreactor body, with an opening on the upper surface of the flask in order for the materials to be placed inside the flask. A voice coil actuator will be attached to the T-75 flask to apply appropriate vibration (changes) to the system, which will be connected to the interior of the flask through drilled holes on the sides of the flask (Titze, 2004). The fibroblast of the ECM will be placed on the simulated ECM structure inside the flask, and submerged in fluid that is similar to the fluid at the larynx. Another function of the fluid is to provide necessary nutrition to the cell for cell culturing. The variable that will be applied to this system would be the vibration caused by the voice coil actuator. As the vibration is continuously applied to the fibroblasts, the alterations of the fibroblast will be observed (Webb, 2004). This will be a closed system, where no air or other molecules come in contact with the culturing fibroblast. Vocal Fold Fibroblasts: Vocal folds (Figure 1), also known as vocal cords, are two pieces of stretchable structure located inside the throat. This structure mainly consists of mucous membranes. There is also a layer of extra-cellular matrix (ECM), which is a “filamentous structure attached to the cell surface and provides traction and positional recognition to the cell” (Titze, 2004). The type of cell that synthesizes and maintains the ECM is called a fibroblast. The fibroblast provides a structural framework for tissues and is also critical in wound healing. Moreover, vocal pathologies are often determined by the conditions of the ECM. These conditions are often due to the alterations, which affect the mechanical 3properties of the tissue between the epithelium and the muscles of the vocal folds (Titze, 2004). Furthermore, there is exists cartilaginous tissue around the vocal folds that controls the vibration and stretching of the vocal folds. The vibration of human vocals folds could naturally occur at the frequency ranging from 100-1000Hz, at and amplitude of 1mm (Titze, 2004). However, the frequency that they mostly vibrate at is a range from 0-400Hz. The vocal folds are horizontally stretched across the larynx. Due to the vibration of the vocal folds, the larynx is exposed to high inertial stress (Titze, 2004). The stretching and relaxing of these two membranes determine the pitch of one’s phonation. The two vocal folds come in contact and create a “mucosal wave”, which produces sound and also allows the exiting of air from the lungs (Altman, 2002). Figure 1. The left figure indicates the diagram of the vocal folds, with parts of the vocal folds labeled. The right figure is an actual screen shot of the vocal folds. Notice the two mucous membranes with the opening in the middle. Problem Motivation: The focus of this project is to construct a working prototype of a bioreactor that would be able to culture vocal fibroblasts for research purposes by subjecting the cell- 4seeded matrices (attached to Tecoflex strips) to a specific range of stimuli. Such stimuli include both ‘violent’ vibrations and tensile stress that would effectively mimic the human vocal fold environment when vibrations (sounds) are produced in the larynx. Research will be conducted while the environment of the bioreactor will be maintained and monitored by software interface, the Labview software in particular. For best results, the distribution of uniform vibrations to the cell-seeded strips would be imperative to the success of a working bioreactor. Problem Statement: The main goal of this design project is to improve and reproduce the current design for the vocal fold bioreactor. The current design does a decent job of culturing vocal fold fibroblasts while promoting characteristic elongation and creation of an extracellular matrix through tensile and vibrational stimuli (Titze et al. 1527). However, there are a few things that could use some improvement. First, there is no contact between the vibrating strips. To more accurately recreate the vocal fold environment, a third ‘contact’ stimulus will be included in addition to the tension and vibration produced by the bioreactor. Second, there is an uneven distribution of vibration along the cell-seeded strips. The single-sided vibration causes more vibrational strain to be exerted nearest the source, and different ideas will be experimented with to more evenly distribute the vibration along these strips. Finally, the strips themselves are made of Tecoflex, which is a porous substrate. Unlike real vocal folds which are lined with smooth, elongated cells, Tecoflex looks like a sponge under a microscope and makes cell culturing more difficult
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