Search

Fluid-induced shear stresses are involved in the development of cardiovascular tissues. In a tissue engineering framework, this stimulus has also been considered as a mechanical regulator of stem cell differentiation. We recently demonstrated that the fluid-oscillating effect in combination with a physiologically-relevant shear stress magnitude contributes to the formation of stem cell-derived de novo heart valve tissues. However, the range of oscillations necessary to induce favorable gene expression and engineered tissue formation is unknown. In this study, we took a computational approach to establish a range of oscillatory shear stresses that may optimize in vitro valvular tissue growth. Taking a biomimetic approach, three physiologically-relevant flow waveforms from the human: (i) aorta, (ii) pulmonary artery and (iii) superior vena cava were utilized to simulate pulsatile flow conditions within a bioreactor that housed 3 tissue specimens. Results were compared to non-physiological pulsatile flow (NPPF) and cyclic flexure-steady flow (Flex-Flow) conditions. The oscillatory shear index (OSI) was used to quantify the fluid-induced oscillations occurring on the specimen surfaces. The range of mean OSI under the physiological conditions investigated was found to be 0.18 ≤ OSI ≤ 0.23. On the other hand, NPPF and Flex-Flow environments yielded a mean OSI of 0.37 and 0.11 respectively, which were 46% higher and 45% lower than physiological conditions. Moreover, we subsequently conducted OSI-based human bone marrow stem cell (HBMSC) culture experiments which resulted in preferential valvular gene expression and phenotype (significant upregulation of BMP, KLF2A, CD31 and α-SMA using an OSI of 0.23 in comparison to a lower OSI of 0.10 or a higher OSI of 0.38; p < .05). These findings suggest that a distinct range or a "sweet-spot" for physiological OSI exists in the mechanical conditioning of tissue engineered heart valves grown from stem cell sources. We conclude that in vitro heart valve matrix development could be further enhanced by simultaneous exposure of the engineered tissues to physiologically-relevant magnitudes of both fluid-induced oscillations and shear stresses., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Engineered valvular tissues are cultured dynamically, and involve specimen movement. We previously demonstrated that oscillatory shear stresses (OSS) under combined steady flow and specimen cyclic flexure (flex-flow) promote tissue formation. However, localized efficiency of specimen mass transport is also important in the context of cell viability within the growing tissues. Here, we investigated the delivery of two essential species for cell survival, glucose and oxygen, to 3-dimensional (3D) engineered valvular tissues. We applied a convective-diffusive model to characterize glucose and oxygen mass transport with and without valve-like specimen flexural movement. We found the mass transport effects for glucose and oxygen to be negligible for scaffold porosities typically present during in vitro experiments and non-essential unless the porosity was unusually low (<40%). For more typical scaffold porosities (75%) however, we found negligible variation in the specimen mass fraction of glucose and oxygen in both non-moving and moving constructs (p > 0.05). Based on this result, we conducted an experiment using bone marrow stem cell (BMSC)-seeded scaffolds under Pulsatile flow-alone states to permit OSS without any specimen movement. BMSC-seeded specimen collagen from the pulsatile flow and flex-flow environments were subsequently found to be comparable (p > 0.05) and exhibited some gene expression similarities. We conclude that a critical magnitude of fluid-induced, OSS created by either pulsatile flow or flex-flow conditions, particularly when the oscillations are physiologically-relevant, is the direct, principal stimulus that promotes engineered valvular tissues and its phenotype, whereas mass transport benefits derived from specimen movement are minimal.

For treatment of critical heart valve diseases, prosthetic valves perform fairly well in most adults; however, for pediatric patients, there is the added requirement that the replacement valve grows with the child, thus extremely limiting current treatment options. Tissue engineered heart valves (TEHV), such as those derived from autologous bone marrow stem cells (BMSCs), have the potential to recapitulate native valve architecture and accommodate somatic growth. However, a fundamental pre-cursor in promoting directed integration with native tissues rather than random, uncontrolled growth requires an understanding of BMSC mechanobiological responses to valve-relevant mechanical environments. Here, we report on the responses of human BMSC-seeded polymer constructs to the valve-relevant stress states of: (i) steady flow alone, (ii) cyclic flexure alone, and (iii) the combination of cyclic flexure and steady flow (flex-flow). BMSCs were seeded onto a PGA: PLLA polymer scaffold and cultured in static culture for 8 days. Subsequently, the aforementioned mechanical conditions, (groups consisting of steady flow alone-850ml/min, cyclic flexure alone-1 Hz, and flex-flow-850ml/min and 1 Hz) were applied for an additional two weeks. We found samples from the flex-flow group exhibited a valve-like distribution of cells that expressed endothelial (preference to the surfaces) and myofibroblast (preference to the intermediate region) phenotypes. We interpret that this was likely due to the presence of both appreciable fluid-induced shear stress magnitudes and oscillatory shear stresses, which were concomitantly imparted onto the samples. These results indicate that flex-flow mechanical environments support directed in vitro differentiation of BMSCs uniquely towards a heart valve phenotype, as evident by cellular distribution and expression of specific gene markers. A priori guidance of BMSC-derived, engineered tissue growth under flex-flow conditions may serve to subsequently promote controlled, engineered to native tissue integration processes in vivo necessary for successful long-term valve remodeling.

Fluid-induced oscillatory shear stress (OSS) and nicotine are known antagonists in cardiovascular disease. However, from a regenerative medicine standpoint, we hypothesized that these parameters may support the cell differentiation of bone marrow mesenchymal stem cells (BMMSCs) for engineering heart valves. In this study, OSS and nicotine (10-6M) were applied individually to BMMSCs in monolayer culture. In both cases, a significantly higher expression of CD31 was detected compared to corresponding controls (p<0.05). We interpret our findings to indicate that both OSS and nicotine independently support mesenchymal to endothelial transformation; however, the underlying mechanism for this transformation in terms of the cell cytoskeletal structure was entirely different between the two stimulants. In the case of OSS, F-actin filaments exhibited a stretching response and formed a preferential alignment with each other. However, in the nicotine-treated group, a clear increase was observed in the number of actin filaments present, which led to the maximum expression of CD31 in comparison to the OSS and control groups. From our findings, we speculate that while nicotine may stimulate an increase in the differentiation of BMMSCs to endothelial cells, OSS may play a greater role in cellular distribution and the eventual creation of a tissue engineered heart valve (TEHV) endothelium.

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 × 10(6) cells/cm(2). 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/cm(2) 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/cm(2); 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.

Previous efforts in heart valve tissue engineering demonstrated that the combined effect of cyclic flexure and steady flow on bone marrow derived stem cell-seeded scaffolds resulted in significant increases in engineered collagen formation [Engelmayr et al. Cyclic flexure and laminar flow synergistically accelerate mesenchymal stem cell-mediated engineered tissue formation: Implications for engineered heart valve tissues. Biomaterials 2006; 27(36): 6083-95]. Here, we provide a new interpretation for the underlying reason for this observed effect. In addition, another related investigation demonstrated the impact of fluid flow on DNA content and quantified the fluid-induced shear stresses on the engineered heart valve tissue specimens [Engelmayr et al. A Novel Flex-Stretch-Flow Bioreactor for the Study of Engineered Heart Valve Tissue Mechanobiology]. Annals of Biomedical Engineering 2008, 36, 1-13]. In this study, we performed more advanced CFD analysis with an emphasis on oscillatory wall shear stresses imparted on specimens when mechanically conditioned by a combination of cyclic flexure and steady flow. Specifically, we hypothesized that the dominant stimulatory regulator of the bone marrow stem cells is fluid-induced and depends on both the magnitude and temporal directionality of surface stresses, i.e., oscillatory shear stresses (OSS) acting on the developing tissues. Therefore, we computationally quantified the (i) magnitude of fluid-induced shear stresses as well as (ii) the extent of temporal fluid oscillations in the flow field using the oscillatory shear index (OSI) parameter. Noting that sample cyclic flexure induces a high degree of OSS, we incorporated moving boundary computational fluid dynamic simulations of samples housed within a bioreactor to consider the effects of: (1) No Flow, No Flexure (control group), (2) Steady Flow-alone, (3) Cyclic Flexure-alone and (4) Combined Steady flow and Cyclic Flexure environments. Indeed we found that the coexistence of both OSS and appreciable shear stress magnitudes explained the high levels of engineered collagen previously observed from combining cyclic flexure and steady flow states. On the other hand, each of these metrics on its own showed no association. This finding suggests that cyclic flexure and steady flow synergistically promote engineered heart valve tissue production via OSS, so long as the oscillations are accompanied by a critical magnitude of shear stress., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Oscillatory shear stress (OSS), caused by time-varying flow environments, may play a critical role in the production of engineered tissue by bone marrow-derived stem cells. This is particularly relevant in heart valve tissue engineering (HVTE), owing to the intense haemodynamic environments that surround native valves. In this study, we examined and quantified the role that (i) physiologically relevant scales of pulsatility and (ii) changes in geometry as a function of specimen flexure have in creating OSS conditions. A U-shaped bioreactor capable of producing flow, stretch and flexure was modelled with housed specimens, and computational fluid dynamic simulations were performed. We found that physiologically relevant OSS can be maximised by the application of pulsatile flow to straight, non-moving specimens in a uniform manner. This finding reduces a substantial layer of complexity in dynamic HVTE protocols in which traditionally, time-varying flow has been promoted through specimen movement in custom-made bioreactors.

In heart valve tissue engineering, assessment of cell migration under dynamic states can provide insights on the evolving tissue structure. We labeled human vascular smooth muscle (SMCs), endothelial (ECs), and bone marrow-derived mesenchymal stem cells (BMSCs) with superparamagnetic iron oxide (SPIO) microparticles and visualized them using magnetic resonance imaging (MRI) under steady flow. We determined that vascular cells were able to remain reasonably viable and proliferate well after being labeled with SPIO microparticles (200 μg/mL) for 48 hours. SPIO-labeled cells were successfully visualized using T2* contrast. When physiologically representative shear stresses (5-6 dynes/cm2) were applied to SMC-EC coculture-seeded scaffolds, hypointense regions seemed to have decreased after 2 weeks in some locations, whereas others revealed sustained levels of T2* contrast; similar observations were seen in the case of BMSC-seeded scaffolds. This could be attributable to increased out-of-plane cell migratory activity, which occurred from the fluid-induced mechanical cues received, which was not previously evidenced in static culture. Vascular cells and BMSCs were labeled with remarkably high concentrations of SPIO. Moreover, steady fluid flow enhanced intrascaffold cell migration of vascular SMCs and ECs as well as BMSCs, which, in turn, significantly improved construct cellularity and extracellular collagen content.

Tissue engineered heart valves (TEHVs) may provide a permanent solution to congenital heart valve disease by permitting somatic valve growth in the pediatric patient. However, to date, TEHV studies have focused primarily on collagen, the dominant component of valve extracellular matrix (ECM). Temporal decreases in other ECM components, such as the glycosaminoglycans (GAGs), generally decrease as cells produce more collagen under mechanically loaded states; nevertheless, GAGs represent a key component of the valve ECM, providing structural stability and hydration to the leaflets. In an effort to retain GAGs within the engineered constructs, here we investigated the utility of the protein fibrin in combination with a valve-like, cyclic flexure and steady flow (flex-flow) mechanical conditioning culture process using adult human periodontal ligament cells (PLCs). We found both fibrin and flex-flow mechanical components to be independently significant (p<0.05), and hence important in influencing the DNA, GAG and collagen contents of the engineered tissues. In addition, the interaction of fibrin with flex-flow was found to be significant in the case of collagen; specifically, the combination of these environments promoted PLC collagen production resulting in a significant difference compared to dynamic and statically cultured specimens without fibrin. Histological examination revealed that the GAGs were retained by fibrin entrapment and adhesion, which were subsequently confirmed by additional experiments on native valve tissues. We conclude that fibrin in the flex-flow culture of engineered heart valve tissues: (i) augments PLC-derived collagen production; and (ii) enhances retention of GAGs within the developing ECM., (Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The intimal and medial linings of the pulmonary artery consist largely of vascular endothelial cells (VECs) and vascular smooth muscle cells (VSMCs), respectively. The migration of these cell types to a potential tissue-engineered pulmonary valve (TEPV) implant process is therefore of interest in understanding the valve remodeling process. Visualization and cell tracking by MRI, which employs hypointense contrast achievable through the use of superparamagnetic iron oxide (SPIO) microparticles to label cells, provides a method in which this can be studied. We investigated the SPIO labeling efficiency of human VECs and VSMCs, and used two- and three-dimensional gradient echo sequences to track the migration of these cells in agar gel constructs. Protamine sulfate (4.5 µg/mL) was used to enhance SPIO uptake and was found to have no influence on cell viability or proliferation. MRI experiments were initially performed using a 9.4-T scanner. The results demonstrated that the spatial positions of hypointense spots were relatively unchanged over 12 days. Subsequent MR experiments performed at 7 T demonstrated that three-dimensional imaging provided the best spatial resolution to assess cell fate. R(2)* maps were bright in SPIO cell-encapsulated gels in comparison with unlabeled counterparts. Signal voids were ruled out as hypointense regions owing to the smooth exponential decay of T(2)* in these voxels. As a next step, we intend to use the SPIO cell labeling and MR protocols established in this study to assess whether hemodynamic stresses will alter the vascular cell migratory patterns. These studies will shed light on the mechanisms of vascular remodeling after TEPV implantation., (Copyright © 2011 John Wiley & Sons, Ltd.)

We have previously shown that combined flexure and flow (CFF) augment engineered heart valve tissue formation using bone marrow-derived mesenchymal stem cells (MSC) seeded on polyglycolic acid (PGA)/poly-L-lactic acid (PLLA) blend nonwoven fibrous scaffolds (Engelmayr, et al., Biomaterials 2006; vol. 27 pp. 6083-95). In the present study, we sought to determine if these phenomena were reproducible at the organ level in a functional tri-leaflet valve. Tissue engineered valve constructs (TEVC) were fabricated using PGA/PLLA nonwoven fibrous scaffolds then seeded with MSCs. Tissue formation rates using both standard and augmented (using basic fibroblast growth factor [bFGF] and ascorbic acid-2-phosphate [AA2P]) media to enhance the overall production of collagen were evaluated, along with their relation to the local fluid flow fields. The resulting TEVCs were statically cultured for 3 weeks, followed by a 3 week dynamic culture period using our organ level bioreactor (Hildebrand et al., ABME, Vol. 32, pp. 1039-49, 2004) under approximated pulmonary artery conditions. Results indicated that supplemented media accelerated collagen formation (approximately 185% increase in collagen mass/MSC compared to standard media), as well as increasing collagen mass production from 3.90 to 4.43 pg/cell/week from 3 to 6 weeks. Using augmented media, dynamic conditioning increased collagen mass production rate from 7.23 to 13.65 pg/cell/week (88.8%) during the dynamic culture period, along with greater preservation of net DNA. Moreover, when compared to our previous CFF study, organ level conditioning increased the collagen production rate from 4.76 to 6.42 pg/cell/week (35%). Newly conducted CFD studies of the CFF specimen flow patterns suggested that oscillatory surface shear stresses were surprisingly similar to a tri-leaflet valve. Overall, we found that the use of simulated pulmonary artery conditions resulted in substantially larger collagen mass production levels and rates found in our earlier CFF study. Moreover, given the fact that the scaffolds underwent modest strains (approximately 7% max) during either CFF or physiological conditioning, the oscillatory surface shear stresses estimated in both studies may play a substantial role in eliciting MSC collagen production in the highly dynamic engineered heart valve fluid mechanical environment., ((c) 2009 Elsevier Ltd. All rights reserved.)