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Research Papers

A Novel Bioreactor for Mechanobiological Studies of Engineered Heart Valve Tissue Formation Under Pulmonary Arterial Physiological Flow Conditions

[+] Author and Article Information
Sharan Ramaswamy

Department of Biomedical Engineering,
Tissue Engineered Mechanics, Imaging and
Materials Laboratory,
College of Engineering and Computing,
Florida International University,
Miami, FL 33174

Steven M. Boronyak

Department of Biomedical Engineering,
Vanderbilt University,
Nashville, TN 37232

Trung Le, Fotis Sotiropoulos

Department of Civil Engineering,
St. Anthony Falls Laboratory,
College of Science and Engineering,
University of Minnesota,
Minneapolis, MN 55414

Andrew Holmes

Swanson School of Engineering,
University of Pittsburgh,
Pittsburgh, PA 15261

Michael S. Sacks

W. A. “Tex” Moncrief, Jr. Simulation-Based
Engineering Science Chair I,
Professor of Biomedical Engineering,
Department of Biomedical Engineering,
Institute for Computational
Engineering and Sciences (ICES),
The University of Texas at Austin,
201 East 24th Street, ACES 5.438,
1 University Station, C0200,
Austin, TX 78712-0027
e-mail: msacks@ices.utexas.edu

1Corresponding author.

Manuscript received July 6, 2013; final manuscript received September 29, 2014; accepted manuscript posted October 16, 2014; published online November 7, 2014. Assoc. Editor: Jonathan Vande Geest.

J Biomech Eng 136(12), 121009 (Dec 01, 2014) (14 pages) Paper No: BIO-13-1301; doi: 10.1115/1.4028815 History: Received July 06, 2013; Revised September 29, 2014; Accepted October 16, 2014

The ability to replicate physiological hemodynamic conditions during in vitro tissue development has been recognized as an important aspect in the development and in vitro assessment of engineered heart valve tissues. Moreover, we have demonstrated that studies aiming to understand mechanical conditioning require separation of the major heart valve deformation loading modes: flow, stretch, and flexure (FSF) (Sacks et al., 2009, "Bioengineering Challenges for Heart Valve Tissue Engineering," Annu. Rev. Biomed. Eng., 11(1), pp. 289–313). To achieve these goals in a novel bioreactor design, we utilized a cylindrical conduit configuration for the conditioning chamber to allow for higher fluid velocities, translating to higher shear stresses on the in situ tissue specimens while retaining laminar flow conditions. Moving boundary computational fluid dynamic (CFD) simulations were performed to predict the flow field under combined cyclic flexure and steady flow (cyclic-flex-flow) states using various combinations of flow rate, and media viscosity. The device was successfully constructed and tested for incubator housing, gas exchange, and sterility. In addition, we performed a pilot experiment using biodegradable polymer scaffolds seeded with bone marrow derived stem cells (BMSCs) at a seeding density of 5 × 106 cells/cm2. The constructs were subjected to combined cyclic flexure (1 Hz frequency) and steady flow (Re = 1376; flow rate of 1.06 l/min (LPM); shear stress in the range of 0–9 dynes/cm2) for 2 weeks to permit physiological shear stress conditions. Assays revealed significantly (P < 0.05) higher amounts of collagen (2051 ± 256 μg/g) at the end of 2 weeks in comparison to similar experiments previously conducted in our laboratory but performed at subphysiological levels of shear stress (<2 dynes/cm2; Engelmayr et al., 2006, "Cyclic Flexure and Laminar Flow Synergistically Accelerate Mesenchymal Stem Cell-Mediated Engineered Tissue Formation: Implications for Engineered Heart Valve Tissues," Biomaterials, 27(36), pp. 6083–6095). The implications of this novel design are that fully coupled or decoupled physiological flow, flexure, and stretch modes of engineered tissue conditioning investigations can be readily accomplished with the inclusion of this device in experimental protocols on engineered heart valve tissue formation.

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Figures

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Fig. 1

Photo of the overall FSF bioreactor device, showing the four separate chambers, associated pump, tubing and culture media changing system. Key components are labeled as shown.

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Fig. 2

(a) Cut-out longitudinal section of the FSF bioreactor showing several key dimensions, (b) close-up view of key components in one of the bioreactor's conditioning chamber. In (c) is a cross section showing orientation and the placement of a specimen when flat in the flow tube, which were placed off-center by a distance of 4.3 mm. When flexed, the specimen will protrude into the center of the tube, moving in the left to right direction. Legend is as follows: A. ULTEM chamber, B. U-shaped fluid enclosure, C. Sliding sample holder, D. Outer tube, E. Ring, F. Moving post, G. Fixed post, and H. Injection port.

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Fig. 3

(a) Viscosity versus shear rate measurements for control (regular) cell culture media and increased-viscosity media, augmented with Xanthan Gum

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Fig. 4

(a) The bioreactor geometry used for CFD modeling. (b) Triangular elements used to mesh the rectangular specimens. (c) Position of the moving post (“b” in Eq. (3)) for the leaflet deformation over one cycle T = 1 s; for completeness, the variation of the quadratic coefficient (“C” in Eq. (3)) with time is also shown. (d) Deformation of the specimens over one cycle, noting that the deforming shape was assumed to take on parabolic profile according to Eq. (3). (e) Location of the specimen inner and outer surfaces.

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Fig. 5

5-h variation of (a) pH, (b) pCO2,  and (c) pO2 levels after placement in incubator

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Fig. 6

Contour of velocity magnitudes in the plane (x = 17.5 mm) at the centerline of the specimens with different Re numbers at t = 0.5 s. Note that because we carried out the numerical simulations over a relatively wide range of Reynolds number from Re = 246 to Re = 1376, there will be a significant difference in velocity magnitude among the bulk flow of the cases, u = 0.07 m/s to u = 0.1333 m/s. and in turn, the magnitude of velocities in the vicinity of the samples will be substantially different. On the other hand, our reason for choosing u = 0.25 m/s as the “cut-off” level was purely for visualization (color scale) purposes so that the flow structure could be compared between cases.

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Fig. 7

Contour of velocity magnitudes in the plane (X = 17.5 mm) at the center line of the leaflet at different time instants during one cycle t = 0, 0.25, 0.5, and 0.75 s of case A (Re = 1376) depicting increasing degrees of specimen flexure. High velocity streaks were augmented with an increase in specimen flexure. Note that because we carried out the numerical simulations over a relatively wide range of Reynolds number from Re = 246 to Re = 1376, there will be a significant difference in velocity magnitude among the bulk flow of the cases, u = 0.07 m/s to u = 0.1333 m/s and in turn, the magnitude of velocities in the vicinity of the samples will be substantially different. On the other hand, our reason for choosing u = 0.25 m/s as the cut-off level was purely for visualization (color scale) purposes so that the flow structure could be compared between cases.

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Fig. 8

Time-averaged specimen shear stress magnitudes and streamlines over one cycle. The following axial locations (Y/D): − 3.5 (specimen 1), −6.34 (specimen 2), −9.2 (specimen 3), corresponding to the center of each specimen, was where the largest magnitude and variation in shear stress magnitude occurred. The outer wall mean shear stress (dynes/cm2) for specimens 1, 2, 3 were 5.5, 7.6, and 7.4, whereas the corresponding inner wall mean shear stress (dynes/cm2) for specimens 1, 2, and 3 were: 2.7, 2.5, and 2.4. These results showed that the average shear stress magnitude was lower for specimen 1 in comparison to specimens 2 and 3 on the outer surface; nonetheless, the difference was comparably small (less than 10%). For the inner wall, the three specimens were subjected to nearly the same value of average shear stress magnitude.

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Fig. 9

Flow patterns around the center specimen for case A (Re = 1376) during one cycle, at the peak flexure state (t = 0.5 s). The velocity vectors were projected on the plane at X = 17.5 mm (i.e., on the centerline of the leaflets). The high velocity streaks were found to be surrounding the sample outer wall, whereas flow vortex formation and reversal occurred proximal to the inner wall, near to the fixed postlocation. The velocity field was considerably lower in the region surrounding the inner wall of the samples. Note that reference positions proximal to the fixed and moving posts have been labeled as A and B in the figure, respectively.

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Fig. 10

Fluid-induced shear stress distribution on the outer wall surface of the intermediate specimen for case A (Re = 1376) during one cycle at t = 0, 0.25, 0.5, and 0.75 s

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Fig. 11

Fluid-induced shear stress distribution on the inner wall surface of the intermediate specimen for case A (Re = 1376) during one cycle at t = 0, 0.25, 0.5, and 0.75 s

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Fig. 12

OSI distribution on the outer (top) and inner (bottom) wall of the three bioreactor specimens for case A (Re = 1376). The streamlines represent the mean shear stress pattern on the sample surface. As seen, OSI values were clearly much higher on the sample inner wall suggestive of utilization of the bioreactor device for cell mechanobiology studies involving oscillatory shear stress. Note that reference positions proximal to the fixed and moving post have been labeled as A and B in the figure, respectively.

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