Stretch Bioreactor Research Articles

Tracheal reconstruction with pedicled tandem grafts engineered by a radial stretch bioreactor.

The engineering of tracheal substitutes is pivotal in improving tracheal reconstruction. In this study, we aimed to investigate the effects of biomechanical stimulation on tissue engineering tracheal cartilage by mimicking the trachea motion through a novel radial stretching bioreactor, which enables to dynamically change the diameter of the hollow cylindrical implants. Applying our bioreactor, we demonstrated that chondrocytes seeded on the surface of Poly (ε-caprolactone) scaffold respond to mechanical stimulation by improvement of infiltration into implants and upregulation of cartilage-specific genes. Further, the mechanical stimulation enhanced the accumulation of cartilage neo-tissues and cartilage-specific extracellular macromolecules in the muscle flap-remodeled implants and reconstructed trachea. Nevertheless, the invasion of fibrous tissues in the reconstructed trachea was suppressed upon mechanical loading.

Compact and tunable stretch bioreactor advancing tissue engineering implementation. Application to engineered cardiac constructs

Physical stimuli are crucial for the structural and functional maturation of tissues both in vivo and in vitro. In tissue engineering applications, bioreactors have become fundamental and effective tools for providing biomimetic culture conditions that recapitulate the native physical stimuli. In addition, bioreactors play a key role in assuring strict control, automation, and standardization in the production process of cell-based products for future clinical application. In this study, a compact, easy-to-use, tunable stretch bioreactor is proposed. Based on customizable and low-cost technological solutions, the bioreactor was designed for providing tunable mechanical stretch for biomimetic dynamic culture of different engineered tissues. In-house validation tests demonstrated the accuracy and repeatability of the imposed mechanical stimulation. Proof of concepts biological tests performed on engineered cardiac constructs, based on decellularized human skin scaffolds seeded with human cardiac progenitor cells, confirmed the bioreactor Good Laboratory Practice compliance and ease of use, and the effectiveness of the delivered cyclic stretch stimulation on the cardiac construct maturation.

A study of extracellular matrix remodeling in aortic heart valves using a novel biaxial stretch bioreactor.

In aortic valves, biaxial cyclic stretch is known to modulate cell differentiation, extracellular matrix (ECM) synthesis and organization. We designed a novel bioreactor that can apply independent and precise stretch along radial and circumferential directions in a tissue culture environment. While this bioreactor can be used for either native or engineered tissues, this study determined matrix remodeling and strain distribution of aortic cusps after culturing under biaxial stretch for 14 days. The contents of collagen and glycosaminoglycans were determined using standard biochemical assays and compared with fresh controls. Strain fields in static cusps were more uniform than those in stretched cusps, which indicated degradation of the ECM fibers. The glycosaminoglycan content was significantly elevated in the static control as compared to fresh or stretched cusps, but no difference was observed in collagen content among the groups. The strain profile of freshly isolated fibrosa vs. ventricularis and left, right, and noncoronary cusps were also determined by Digital Image Correlation technique. Distinct strain patterns were observed under stretch on fibrosa and ventricularis sides and among the three cusps. This work highlights the critical role of the anisotropic ECM structure for proper functions of native aortic valves and the beneficial effects of biaxial stretch for maintenance of the native ECM structure.

Pulsatile flow conditioning of three-dimensional bioengineered cardiac ventricle

Current physical stimuli mechanical stretch bioreactor studies focus on conditioning planar and/or tubular engineered cardiac constructs. The current 3D bioreactor models in cardiac tissue engineering use differential pressure loading for structural support as opposed to conditioning. The development of the pulsatile flow conditioned ventricle (PFCV) provides a 3D mechanical stretch conditioning method to generate pump function in the engineered cardiac left ventricle. The study utilizes a chitosan bioengineered open ventricle scaffold, to produce the in vitro PFCV model. PFCV were fabricated by wrapping the outer scaffold surface with a 3D fibrin gel artificial heart muscle patch, followed by pulsatile flow conditioning for 20 h. The average contractile frequency was 57 bpm. The average pressure generated, under maintained flow, post-conditioning, was 3.1633 mmHg. The average biopotential output was 0.4881 mV. Histologically, the PFCV displayed a more disseminated presence of intercellular interactions and sarcomeric organization. The results of this study clearly demonstrate the effectiveness of pulsatile flow conditioning to improve the function of our engineered left ventricle.

Conditioning of Cardiovascular Tissue Using a Noncontact Magnetic Stretch Bioreactor with Embedded Magnetic Nanoparticles.

Bioreactor systems, an integral component of tissue engineering, are designed to simulate complex in vivo conditions to impart functionality to artificial tissue. All standard forms of stretch bioreactors require physical contact with artificial heart muscle (AHM). However, we believe that noncontact stretch bioreactors have the potential to lead to higher functional benefit of AHM. Our work is focused on the fabrication of a noncontact magnetic stretch bioreactor (MSB) that uses magnetic nanoparticles to simulate stretch conditions to impart functionality. During our development of this system, we applied magnetically induced stretch conditioning through application of an oscillating magnetic field to a ferromagnetic heart muscle model. Fibrin scaffolds were loaded with magnetic nanoparticles prior to tissue model formation. Oscillating magnetic fields were applied by a novel bioreactor system through displacement of a neodymium magnet. The addition of commercially obtained iron(III) oxide (Fe2O3) in sufficient quantities to allow for physiologically relevant stretches (15% axial displacement) caused toxic effects after 4 days of culture. In contrast, loading scaffolds with monodispersed, high-saturation-magnetization magnetite (Fe3O4) nanoparticles specifically prepared for these experiments increased the field strength of the magnetized fibrin 10-fold over polydispersed, low-saturation magnetization, Fe2O3. Additionally, loading with Fe3O4 enabled magnetically actuated stretching with markedly reduced toxicity over 8 days of culture. Using a 20% stretch 0.5 Hz protocol, we observed a significant increase in twitch force over controls at days 4 and 6. This work provides a technology for controlled noncontact mechanical stretch to condition AHM.

In vitro assessment of a combined radiofrequency ablation and cryo-anchoring catheter for treatment of mitral valve prolapse

Percutaneous approaches to mitral valve repair are an attractive alternative to surgical repair or replacement. Radiofrequency ablation has the potential to approximate surgical leaflet resection by using resistive heating to reduce leaflet size, and cryogenic temperatures on a percutaneous catheter can potentially be used to reversibly adhere to moving mitral valve leaflets for reliable application of radiofrequency energy. We tested a combined cryo-anchoring and radiofrequency ablation catheter using excised porcine mitral valves placed in a left heart flow loop capable of reproducing physiologic pressure and flow waveforms. Transmitral flow and pressure were monitored during the cryo-anchoring procedure and compared to baseline flow conditions, and the extent of radiofrequency energy delivery to the mitral valve was assessed post-treatment. Long term durability of radiofrequency ablation treatment was assessed using statically treated leaflets placed in a stretch bioreactor for four weeks. Transmitral flow and pressure waveforms were largely unaltered during cryo-anchoring. Parameter fitting to mechanical data from leaflets treated with radiofrequency ablation and cryo-anchoring revealed significant mechanical differences from untreated leaflets, demonstrating successful ablation of mitral valves in a hemodynamic environment. Picrosirius red staining showed clear differences in morphology and collagen birefringence between treated and untreated leaflets. The durability study indicated that statically treated leaflets did not significantly change size or mechanics over four weeks. A cryo-anchoring and radiofrequency ablation catheter can adhere to and ablate mitral valve leaflets in a physiologic hemodynamic environment, providing a possible percutaneous alternative to surgical leaflet resection of mitral valve tissue.

Creation of a bioreactor for the application of variable amplitude mechanical stimulation of fibrin gel-based engineered cardiac tissue.

This chapter details the creation of three-dimensional fibrin hydrogels as an engineered myocardial tissue and introduces a mechanical stretch bioreactor system that allows for the cycle-to-cycle variable amplitude mechanical stretch of the constructs as a method of conditioning the constructs to be more similar to native tissue. Though mechanical stimulation has been established as a standard method of improving construct development, most studies have been performed under constant frequency and constant amplitude, even though variability is a critical aspect of healthy cardiac physiology. The introduction of variability in other organ systems has demonstrated beneficial effects to cell function in vitro. We hypothesize that the introduction of variability in engineered cardiac tissue could have a similar effect.

Abstract 452: Cyclic Strain Induces Osteopontin Expression in Vascular Smooth Muscle via a Hydrogen Peroxide--Dependent Mechanism

Introduction: Vascular inflammation, a hallmark of hypertension, is associated with increased cyclic strain on the vessel wall. Reactive oxygen species (ROS), such as superoxide and hydrogen peroxide, are known to play an important role in the progression of cardiovascular inflammation. However, the underlying molecular mechanisms of ROS modulation of vascular inflammation through increased cyclic strain remain largely unknown. Objective: In this study, we sought to determine whether cyclical strain induces an increase in osteopontin (OPN), a pro-inflammatory protein, in a hydrogen peroxide dependent manner using an in vitro cell system. Methods: Rat aortic smooth muscle cells were seeded on collagen-I coated, flexible bottomed six-well plates and cyclically stretched at 10% elongation in a biaxial stretch bioreactor. Following stretch, cells were harvested and protein, mRNA, and hydrogen peroxide levels were measured via Western Blotting, quantitative real-time PCR, and Amplex Red assay, respectively. Results: Preliminary results indicate that cyclic strain promotes a 16% ± 6 increase in hydrogen peroxide levels compared to non-stretched cells at 8 hours. In addition, elevated expression of OPN protein was observed with 24 hours of stretch (195% ± 16). Increased OPN mRNA was also observed with 12 hours of stretch (39% ± 15). Finally, OPN expression was blunted when cells were simultaneously stretched for 24 hours with 200U/mL PEG-Catalase (a H2O2 scavenger) suggesting that the stretch induced increase in OPN expression is mediated by hydrogen peroxide (49%±14 decrease in cells stretched with PEG-Catalase versus non- PEG-Catalase treated but stretched cells) Conclusion: These data support that increased cyclical mechanical stretch, as experienced by the vascular wall under hypertensive conditions, increases osteopontin expression, via a hydrogen peroxide-mediated pathway.

Aortic Valve Cyclic Stretch Causes Increased Remodeling Activity and Enhanced Serotonin Receptor Responsiveness

Increased serotonin (5-hydroxytryptamine [5HT]) receptor (5HTR) signaling has been associated with cardiac valvulopathy. Prior cell culture studies of 5HTR signaling in heart valve interstitial cells have provided mechanistic insights concerning only static conditions. We investigated the hypothesis that aortic valve biomechanics participate in the regulation of both 5HTR expression and interrelated extracellular matrix remodeling events. The effects of cyclic stretch on aortic valve 5HTR, expression, signaling, and extracellular matrix remodeling were investigated using a tensile stretch bioreactor in studies which also compared the effects of adding 5HT and (or) the 5HT-transporter inhibitor, fluoxetine. Cyclic stretch alone increased both proliferation and collagen in porcine aortic valve cusp samples. However, with cyclic stretch, unlike static conditions, 5HT plus fluoxetine caused the greatest increase in proliferation (p<0.0001), and also caused significant increases in collagen (p<0.0001) and glycosaminoglycans (p<0.0001). The DNA microarray data demonstrated upregulation of 5HTR2A and 5HTR2B (>4.5-fold) for cyclic stretch versus static (p<0.001), while expression of the 5HT transporter was not changed significantly. Extracellular matrix genes (eg, collagen types I, II, III, and proteoglycans) were also upregulated by cyclic stretch. Porcine aortic valve cusp samples subjected to cyclic stretch upregulate 5HTR2A and 2B, and also initiate remodeling activity characterized by increased proliferation and collagen production. Importantly, enhanced 5HTR responsiveness due to increased 5HTR2A and 2B expression results in a significantly greater response in remodeling endpoints (proliferation, collagen, and GAG production) to 5HT in the presence of 5HT transporter blockade.

Elevated Cyclic Stretch Induces Aortic Valve Calcification in a Bone Morphogenic Protein-Dependent Manner

Calcified aortic valve (AV) cusps have increased expression of bone morphogenic proteins (BMPs) and transforming growth factor-beta1 (TGF-beta1). Elevated stretch loading on the AV is known to increase expression of matrix remodeling enzymes and pro-inflammatory proteins. Little, however, is known about the mechanism by which elevated stretch might induce AV calcification. We investigated the hypothesis that elevated stretch may cause valve calcification via a BMP-dependent mechanism. Porcine AV cusps were cultured in a stretch bioreactor, at 10% (physiological) or 15% (pathological) stretch and 70 beats per minute for 3, 7, and 14 days, in osteogenic media supplemented with or without high phosphate (3.8 mmol/L), TGF-beta1 (1 ng/ml), as well as the BMP inhibitor noggin (1, 10, and 100 ng/ml). Fresh cusps served as controls. Alizarin red and von Kossa staining demonstrated that 15% stretch elicited a stronger calcification response compared with 10% stretch in a fully osteogenic medium containing high phosphate and TGF-beta1. BMP-2, -4, and Runx2 expression was observed after 3 days on the fibrosa surface of the valve cusp and was stretch magnitude-dependent. Cellular apoptosis was highest at 15% stretch. Tissue calcium content and alkaline phosphatase activity were similarly stretch-dependent and were significantly reduced by noggin in a dose dependent manner. These results underline the potential role of BMPs in valve calcification due to altered stretch.

Controlled cyclic stretch bioreactor for tissue-engineered heart valves

A tissue-engineered heart valve (TEHV) represents the ultimate valve replacement, especially for juvenile patients given its growth potential. To date, most TEHV bioreactors have been developed based on pulsed flow of culture medium through the valve lumen to induce strain in the leaflets. Using a strategy for controlled cyclic stretching of tubular constructs reported previously, we developed a controlled cyclic stretch bioreactor for TEHVs that leads to improved tensile and compositional properties. The TEHV is mounted inside a latex tube, which is then cyclically pressurized with culture medium. The root and leaflets stretch commensurately with the latex, the stretching being dictated by the stiffer latex and thus controllable. Medium is also perfused through the lumen at a slow rate in a flow loop to provide nutrient delivery. Fibrin-based TEHVs prepared with human dermal fibroblasts were subjected to three weeks of cyclic stretching with incrementally increasing strain amplitude. The TEHV possessed the tensile stiffness and stiffness anisotropy of leaflets from sheep pulmonary valves and could withstand cyclic pulmonary pressures with similar distension as for a sheep pulmonary artery.

Elevated cyclic stretch alters matrix remodeling in aortic valve cusps: implications for degenerative aortic valve disease

Matrix metalloproteinases (MMPs) and cathepsins are proteolytic enzymes that are upregulated in diseased aortic valve cusps. The objective of this study was to investigate whether elevated cyclic stretch causes an increased expression and activity of these proteolytic enzymes in the valve cusp. Circumferentially oriented fresh porcine aortic valve cusp sections were stretched to 10% (physiological), 15% (pathological), and 20% (hyperpathological) in a tensile stretch bioreactor for 24 and 48 h. The expression and activity of MMP-1, MMP-2, MMP-9, tissue inhibitor of MMP-1, and cathepsin L, S, and K were quantified and compared with fresh controls. Cell proliferation and apoptosis were also analyzed. As a result, at 10% physiological stretch, the expression and activity of remodeling enzymes were comparable with fresh controls. At 15% stretch, the expression of MMP-1, -2, -9 and cathepsin S and K were upregulated, whereas the expression of cathepsin L was downregulated compared with controls. A similar trend was observed at 20% stretch, but the magnitudes of upregulation and downregulation of the expression were less than those observed at 15%. In addition, there were significantly higher cell proliferation and apoptosis at 20% stretch compared with those of other treatment groups. In conclusion, elevated mechanical stretch on aortic valve cusps may detrimentally alter the proteolytic enzyme expression and activity in valve cells. This may trigger a cascade of events leading to an accelerated valve degeneration and disease progression.