Name: a multi-cue bioreactor to evaluate the inflammatory and regenerative capacity of biomaterials under flow and stretch
Uploaded: 2020-12-10T13:00:00.000+00:00
Duration: 7 min 51 s
Description: Watch this Scientific Journal Video about A Multi-Cue Bioreactor to Evaluate the Inflammatory and Regenerative Capacity of Biomaterials under Flow and Stretch at JoVE.com

This study is financially supported by ZonMw as part of the LSH 2Treat program (436001003) and the Dutch Kidney Foundation (14a2d507). N.A.K. acknowledges support from the European Research Council (851960). We gratefully acknowledge the Gravitation Program &#8220;Materials Driven Regeneration&#8221;, funded by the Netherlands Organization for Scientific Research (024.003.013).

The authors have nothing to disclose.

The bioreactor described herein allows for the systematic evaluation of the contributions of the individual and combined effects of shear stress and cyclic stretch on inflammation and tissue regeneration in tubular resorbable scaffolds. This approach also enables a large variety of analyses to be performed on vascular constructs, as exemplified in the representative results section. These results show the distinctive impact of the different hemodynamic loading regimes (i.e., different combinations of shear and stretch) o...

Cardiovascular tissue engineering (TE) is being pursued as an alternative treatment option to the currently used permanent cardiovascular prostheses (e.g., vascular grafts, heart valve replacements), which are suboptimal for large cohorts of patients1,2,3,4. Much sought-after applications include tissue-engineered vascular grafts (TEVGs)5,6 and heart valves (TEHVs)7,8. Most often, cardiovascular TE methodologies make use of resorbable biomaterials (either natural or synthetic) that serve as an instructive scaffold for the new tissue to be formed. The formation of new tissue can either be engineered completely in vitro, by seeding the scaffold with cells and culturing in a bioreactor prior to implantation (in vitro TE)9,10,11, or directly in situ, in which the synthetic scaffold is implanted without pre-culturing in order to induce the formation of new tissue directly in the body (in situ TE)12,13,14. For both in vitro and in situ cardiovascular TE approaches, successful functional regeneration is dominantly dependent on both the host immune response to the implanted construct and appropriate biomechanical loading.
The importance of biomechanical loading for cardiovascular TE is well-acknowledged15. In the case of cardiovascular implants, the cells that populate the scaffold are exposed to cyclic stretch and shear stresses that arise as a result of the hemodynamic environment. Numerous studies have reported the stimulatory effect of (cyclic) stretch on the formation of matrix components, such as collagen16,17,18,19, glycosaminoglycans (GAGs)20, and elastin21,22, by various cell types. For example, Huang et al. demonstrated that biaxial stretch elevated the deposition and organization of collagen and elastin in in vitro TEVGs by using a vascular bioreactor23. While the emphasis typically lies on stretch as the dominant load, these studies often make use of flow-driven bioreactors in which the sample is also exposed to shear flow. Although relatively little is known about the isolated influence of shear stresses on tissue formation and inflammation in 3D, some data are available. For example, Hinderer et al. and Eoh et al. demonstrated that shear flow, in addition to a 3D scaffold microstructure, was important for the formation of mature elastin by human vascular smooth muscle cells in an in vitro model system24,25. Altogether, these findings illustrate the relevance of both cyclic stretch and shear stress for cardiovascular TE.
Another important determinant for the success or failure of TE implants is the host&#8217;s immune response to the implanted graft26. This is particularly important for material-driven in situ TE strategies, which actually rely on the acute inflammatory response to the scaffold to kickstart the subsequent processes of cellular influx and endogenous tissue formation and remodeling27. The macrophage is a critical initiator of functional tissue regeneration, which has been shown by multiple studies28,29,30. Analogous to wound healing, the regeneration of tissue is governed by paracrine signaling between macrophages and tissue-producing cells such as fibroblasts and myofibroblasts31,32,33. In addition to coordinating new tissue deposition, macrophages are involved in the active resorption of foreign scaffold material34,35. As such, the in vitro macrophage response to a biomaterial has been identified as a predictive parameter for the in vivo success of implants36,37,38.
The macrophage response to an implanted scaffold is dependent on scaffold design features such as material composition and microstructure35,39,40. In addition to scaffold properties, the macrophage response to a scaffold and their crosstalk with myofibroblasts is also impacted by hemodynamic loads. For example, cyclic stretch was shown to be an important modulator of macrophage phenotype41,42,43,44 and the secretion of cytokines43,44,45,46 in 3D electrospun scaffolds. Using a co-culture system of macrophages and vascular smooth muscle cells, Battiston et al. demonstrated that the presence of macrophages led to increased levels of elastin and GAGs and that moderate levels of cyclic stretch (1.07&#8211;1.10) stimulated the deposition of collagen I and elastin47. In previous works, we have&#160;demonstrated that shear stress is an important determinant for monocyte recruitment into 3D electrospun scaffolds48,49, and that both shear stress and cyclic stretch impact the paracrine signaling between human monocytes and mesenchymal stromal cells50. Fahy et al. demonstrated that shear flow increased the secretion of pro-inflammatory cytokines by human monocytes51.
Taken together, the above evidence shows that an adequate understanding of and control over hemodynamic loads is crucial for cardiovascular TE, and that it is important to consider the inflammatory response to achieve this. Numerous bioreactors have been described previously for the in vitro52,53,54,55,56,57,58 or ex vivo59,60,61 culture of cardiovascular tissues. However, all these systems are designed to mimic the physiological hemodynamic loading conditions as much as possible. While this is highly valuable for the purpose of creating cardiovascular tissues in vitro or maintaining ex vivo cultures, such systems do not allow for systematic studies into the individual effects of individual cues. This is because the application of both cyclic stretch and shear stress in these bioreactors is driven by the same pressurized flow, which intrinsically links them. While microsystems that allow for accurate multi-cue mechanical manipulation have been described for 2D substrates62 or 3D hydrogel setups63,64, such setups do not allow for the incorporation of elastomeric 3D biomaterial scaffolds.
Here, we present the application of a tubular bioreactor system that uniquely enables the decoupling of shear stress and cyclic stretch and helps to mechanistically investigate their individual and combined effects. This system allows for testing of a broad variety of tissue engineered vascular grafts (e.g., synthetic or natural origin, different micro-architecture, various porosities). To effectively decouple the application of shear stress and stretch, the key concepts that the bioreactor uses are (1) separation of the control of shear stress and stretch using distinct pump systems and (2) stimulation of the scaffolds in an &#8216;inside-out&#8217; manner with computationally driven dimensions. Flow is applied on the outside surface of the tubular scaffold through the use of a flow pump, whereas circumferential stretch of the scaffold is induced by expanding a silicone tube on which the scaffold is mounted through the use of a separate strain pump. The dimensions of the silicone tube and the glass tube that contains the construct are carefully chosen and validated using computational fluid dynamics simulations, to ensure that the shear stress on the scaffold (due to flow) and the circumferential stretch (due to tube expansion) do not significantly affect each other. This inside-out design has several practical rationales. If stretch is applied by the luminal fluid pressure (similar to physiological loading), it&#160;inherently requires the sample design to be leak-free. In addition, the pressure required to stretch the sample would be completely determined by the sample stiffness, which may vary between samples and within a sample over time, making it difficult to control the stretch. This bioreactor mounts the tissue engineered graft around a silicone tube and allows for wall shear stress (WSS) application on the outer wall of the graft and pressurizes the graft from the inside. This way, equal loading conditions between samples and within samples over time can be ensured, and moreover, the samples are allowed to be leaky, as is common for porous vascular scaffolds19. This inside-out bioreactor is specifically intended for systematic studies on the effects of shear and/or stretch, rather than the engineering of a native-like blood vessel in vitro, for which traditional vascular bioreactor setups are more suitable. See Figure 1A&#8211;B for the bioreactor design drawings, and its corresponding Table 1 for a functional description and rationale behind the main components of the bioreactor.
The use of the bioreactor is demonstrated on the basis&#160;of a series of recent studies by our group in which we investigated the individual and combined influences of shear stress and cyclic stretch on inflammation and tissue formation in resorbable electrospun scaffolds for in situ cardiovascular tissue19,43,44. To that end, we used human macrophages and myofibroblasts either in mono- or in co-culture to simulate the various phases of the in situ regenerative cascade. We have&#160;demonstrated that cytokine secretion by human macrophages is distinctly impacted by both cyclic stretch and shear stress, affecting the matrix deposition and organization by human myofibroblasts in these scaffolds, both via paracrine signaling and direct contact19,43,44. Notably, these studies revealed that in the case of combined application of shear stress and stretch, the effects on tissue formation and inflammation are either dominated by one of the two loads, or there are synergistic effects of both loads. These findings illustrate the relevance of decoupling both loads to gain a better understanding of the contribution of the mechanical environment on TE processes. This understanding can be applied to systematically optimize scaffold design parameters in relevant hemodynamic loading regimes. In addition, the mechanistic data from such well-controlled environments may serve as input for numerical models that are being developed to predict the course of in situ tissue remodeling, as recently reported for TEVGs65 or TEHVs66, to further improve predictive capacity.

<table><tbody><tr><td>advanced Dulbecco&amp;rsquo;s modified EagleMedium (aDMEM)</td><td>Gibco</td><td>12491-015</td><td>cell culture medium for fibroblasts</td></tr><tr><td>Aqua Stabil</td><td>Julabo</td><td>8940012</td><td>prevent microorganism growth in bioreactor-hydraulic reservoir</td></tr><tr><td>Bovine fibrinogen</td><td>Sigma</td><td>F8630</td><td>to prepare fibrinogen gel to seed the cells on the electrospun scaffold</td></tr><tr><td>Bovine thrombin</td><td>Sigma</td><td>T4648</td><td>to prepare fibrinogen gel to seed the cells on the electrospun scaffold</td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5804</td><td>to spin down cells and conditioned medium</td></tr><tr><td>Clamp scissor - &quot;kelly forceps&quot;</td><td>Almedic</td><td>P-422</td><td>clamp the silicone tubing and apply pre-stretch to the scaffold so the scaffold can be sutured into the engraved groove (autoclave at step 1, step 7)</td></tr><tr><td>CO2 cell culture incubators</td><td>Sanyo</td><td>MCO-170AIC-PE</td><td>for cell culturing</td></tr><tr><td>Compressed air reservoir</td><td>Festo</td><td>CRVZS-5</td><td>smoothing air pressure fluctuations and create time delays for pressure build-up</td></tr><tr><td>Custom Matlab script to calculate the maximum stretches</td><td>Matlab</td><td>R2017. The Mathworks, Natick, MA</td><td>calculate the minimum and maximum outer diameter of the electrospun scaffold</td></tr><tr><td>Data acquisition board</td><td>National Instruments</td><td>BNC-2090</td><td>data processing in between amplifier system and computer</td></tr><tr><td>Ethanol</td><td>VWR</td><td>VWRK4096-9005</td><td>to keep sterile working conditions</td></tr><tr><td>Fetal bovine calf serum (FBS)</td><td>Greiner</td><td>758087</td><td>cell culture medium supplement; serum-supplement</td></tr><tr><td>Flow culture chamber compartments, consisting of a pressure conduit with engraved grooves and small holes to apply pressure on silicone tubing, a screw thread, nose cone, top compartment with flow inlet and bottom compartment flow outlet, adapter bushing</td><td>Custom made, Department of Biomedical Engineering, Eindhoven University of Technology</td><td>n.a.</td><td>flow culture chamber compartments (autoclave at step 1, step 7)</td></tr><tr><td>Glass Pasteur pipet</td><td>Assistant</td><td>HE40567002</td><td>apply vacuum on electrospun scaffold (autoclave at step 1)</td></tr><tr><td>Glass tubes of the flow culture chamber</td><td>Custon made, Equipment &amp;amp; Prototype Center, Eindhoven University of Technology</td><td>n.a.</td><td>part of the flow culture chamber (clean and store in 70% ethanol, at step 1 and 7)</td></tr><tr><td>GlutaMax</td><td>Gibco</td><td>35050061</td><td>cell culture medium amino acid supplement, minimizes ammonia build-up</td></tr><tr><td>High speed camera</td><td>MotionScope</td><td>M-5</td><td>to monitor the stretch during culture; time-lapse photographs of the scaffolds are captured at a frequency of 30 Hz for 6 sec (i.e. 3 stretch cycles)</td></tr><tr><td>High speed camera lens - Micro-NIKKOR 55mm f/2.8 - lens</td><td>Nikon</td><td>JAA616AB</td><td>to monitor the stretch during culture; time-lapse photographs of the scaffolds are captured at a frequency of 30 Hz for 6 sec (i.e. 3 stretch cycles)</td></tr><tr><td>Hose clip</td><td>ibidi GmbH</td><td>10821</td><td>block medium flow (autoclave at step 1, step 7)</td></tr><tr><td>Hydraulic reservoir with 8 screw threads for 8 flow culture chambers</td><td>Custom made, Department of Biomedical Engineering, Eindhoven University of Technology</td><td>n.a.</td><td>to apply pressure to the silicone mounted constructs (clean outside with a paper tissue with 70% ethanol, rinse reservoir with 70% ethanol followed by demi water, at step 1 and 7)</td></tr><tr><td>Ibidi pump system (8x) including ibidi pump, PumpControl software, fluidic unit, perfusion set (medium tubing), air pressure tubing, drying bottles with orange silica beads</td><td>ibidi GmbH</td><td>10902</td><td>set up used to control the flow in the flow culture chambers. Note 1: the ibidi pumps were modified by the manufacturer to enable 200 mbar capacity. Note 2: can be replaced by pump system of other manufacturer, as long as same flow regimes can be applied.</td></tr><tr><td>Knives (no.10 sterile blades, individual foil pack) and scalpel handle (stainless steel, individually wrapped)</td><td>Swann Morton</td><td>0301; 0933</td><td>to cut the silicone tubing in the correct size for the scaffold and to cut the suture material</td></tr><tr><td>LabVIEW Software</td><td>National Instruments</td><td>version 2018</td><td>to control the stretch applied to the scaffolds</td></tr><tr><td>Laminar flow biosafety cabinet with UV light</td><td>Labconco</td><td>302310001</td><td>to ensure sterile working conditions. The UV is used to decontaminate everything that cannot be autoclaved, or touched after autoclaving</td></tr><tr><td>Large and small petri dishes</td><td>Greiner</td><td>664-160</td><td>for sterile working conditions</td></tr><tr><td>L-ascorbic acid 2-phosphate (vitamin C)</td><td>Sigma</td><td>A8960</td><td>cell culture medium supplement, important for collagen production</td></tr><tr><td>LED light cold source KL2500</td><td>Zeiss</td><td>Schott AG</td><td>to aid in visualization for the time lapse of the scaffolds during monitoring of the stretch</td></tr><tr><td>Luer (female and male) locks and connectors, white luer caps</td><td>ibidi GmbH</td><td>various, see (https://ibidi.com/26-flow-accessories)</td><td>to close or connect parts of the bioreactor and the ibidi pump (autoclave at step 1, step 7)</td></tr><tr><td>Measuring amplifier (PICAS)</td><td>PEEKEL instruments B.V.</td><td>n.a.</td><td>to amplify the signal from the pressure sensor and feedback to LabView</td></tr><tr><td>Medium reservoir (large syringes 60 mL) and reservoir holders</td><td>ibidi GmbH</td><td>10974</td><td>medium reservoir (autoclave at step 1, step 7)</td></tr><tr><td>Medium tubing with 4.25 mm outer diameter and 1 mm inner diameter</td><td>Rubber BV</td><td>1805</td><td>to allow for a larger flow rate, the ibidi medium tubing with larger diameter is used. Note: the part of medium tubing guided through the fluidic unit valves are the same as the default ibidi medium tubing</td></tr><tr><td>Motion Studio Software</td><td>Idtvision</td><td>2.15.00</td><td>to make the high speed time lapse images for stretch monitoring</td></tr><tr><td>Needle (19G)</td><td>BD Microlance</td><td>301700</td><td>together with thin flexible tubing used to fill the hydraulic reservoir with ultrapure water without adding air bubbles</td></tr><tr><td>Needle driver</td><td>Adson</td><td>2429218</td><td>to handle the needle of the nylon suture through the silicone tube (autoclave at step 1, step 7)</td></tr><tr><td>Paper tissues</td><td>Kleenex</td><td>38044001</td><td>for cleaning of the equipment with 70% ethanol</td></tr><tr><td>Parafilm</td><td>Sigma</td><td>P7793-1EA</td><td>quick fix if leakage occurs</td></tr><tr><td>Penicillin/streptomycin (P/S)</td><td>Lonza</td><td>DE17-602E</td><td>cell culture medium supplement; prevent bacterial contamination</td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Sigma</td><td>P4417-100TAB</td><td>for storage and washing steps (autoclave at step 1)</td></tr><tr><td>Plastic containers (60 mL) with red screw caps</td><td>Greiner</td><td>206202</td><td>to prepare the fibrinogen solution</td></tr><tr><td>Pneumatic cylinder</td><td>Festo</td><td>AEVC-20-10-I-P</td><td>to actuate the Teflon bellow (clean with a paper tissue with 70% ethanol at step 1 and 7)</td></tr><tr><td>Polycaprolactone bisurea (PCL-BU) tubular scaffolds (3 mm inner diameter, 200 &amp;micro;m wall thickness, 20 mm length)</td><td>SyMO-Chem, Eindhoven, The Netherlands</td><td>n.a.</td><td>produced using electrospinning from 15% (w/w) chloroform (Sigma; 372978) polymer solutions. See Van Haaften et al Tissue Engineering Part C (2018) for more details</td></tr><tr><td>Pressure conduit without holes (for static control)</td><td>Custom made, Department of Biomedical Engineering, Eindhoven University of Technology</td><td>n.a.</td><td>to mount electrospun tubes on silicon tubing (autoclave at step 1, step 7)</td></tr><tr><td>Pressure sensor and transducer</td><td>BD</td><td>TC-XX and P 10 EZ</td><td>the air pressure going to the pneumatic actuated pump is raised until it reaches the set pressure</td></tr><tr><td>Proportional air pressure control valve and pressure sensor</td><td>Festo</td><td>MPPES-3-1/8-2-010, 159596</td><td>provides compressed air to the pneumatic actuated pump</td></tr><tr><td>Roswell Park Memorial Institute 1640 (RPMI-1640)</td><td>Gibco</td><td>A1049101</td><td>cell culture medium for monocyte/macrophage</td></tr><tr><td>Safe lock Eppendorf tubes (1.5 mL)</td><td>Eppendorf</td><td>30120086</td><td>multiple applications (autoclave at step 1)</td></tr><tr><td>Sodium dodecyl sulfate solution 20%</td><td>Sigma</td><td>5030</td><td>Used to clean materials, at a concentration of 0.1%.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Silicone O-rings</td><td>Technirub</td><td>1250S</td><td>to prevent leakage (autoclave at step 1, step 7)</td></tr><tr><td>Silicone tubing (2.8 mm outer diameter, 400 um wall thickness)</td><td>Rubber BV</td><td>1805</td><td>to mount the electrospun tubes on the pressure conduits (autoclave at step 1)</td></tr><tr><td>Sterile tube (15 mL)</td><td>Falcon</td><td>352095</td><td>multiple applications</td></tr><tr><td>Suture, 5-0 prolene with pre-attached taper point needle</td><td>Ethicon, Johnson&amp;amp;Johnson</td><td>EH7404H</td><td>Prolene suture wire 5-0 (75cm length, TF taper point needle, 1/2 circle, 13 mm needle length)</td></tr><tr><td>Syringe (24 mL)</td><td>B. Braun Melsungen AG</td><td>2057932</td><td>to add the ultrapure water or medium to the hydraulic reservoir or flow culture chamber</td></tr><tr><td>Syringe filter (0.2 &amp;micro;m)</td><td>Satorius</td><td>17597-K</td><td>to filter the fibrinogen solution</td></tr><tr><td>T150 cell culture flask with filter cap</td><td>Nunc</td><td>178983</td><td>to degas culture medium</td></tr><tr><td>T75 Cell culture flask with filter cap</td><td>Nunc</td><td>156499</td><td>to culture static control samples</td></tr><tr><td>Teflon bellow</td><td>Custom made, Department of Biomedical Engineering, Eindhoven University of Technology</td><td>n.a.</td><td>to load the hydraulic reservoir (clean outside with a paper tissue with 70% ethanol at step 1 and 7)</td></tr><tr><td>Tray (stainless steel)</td><td>PolarWare</td><td>15-248</td><td>for easy transport of the fluidic culture chambers and the bioreactor from incubator to laminar flow cabinet and back (clean with a paper tissue with 70% ethanol before and after use)</td></tr><tr><td>Tweezers</td><td>Wironit</td><td>4910</td><td>sterile handling of individual parts (autoclave at step 1 and 7)</td></tr><tr><td>Ultrapure water</td><td>Stakpure</td><td>Omniapure UV 18200002</td><td>to correct for medium evaporation, mixed with aqua stabil mixed and used as hydraulic fluid. (autoclave ultrapure water at step 1)</td></tr><tr><td>UV light</td><td>Philips</td><td>TUV 30W/G30 T8</td><td>for decontamination of grafts and bioreactor parts before seeding</td></tr></tbody></table>

a multi-cue bioreactor to evaluate the inflammatory and regenerative capacity of biomaterials under flow and stretch

The use of resorbable biomaterials to induce regeneration directly in the body is an attractive strategy from a translational perspective. Such materials induce an inflammatory response upon implantation, which is the driver of subsequent resorption of the material and the regeneration of new tissue. This strategy, also known as in situ tissue engineering, is pursued to obtain cardiovascular replacements such as tissue-engineered vascular grafts. Both the inflammatory and the regenerative processes are determined by the local biomechanical cues on the scaffold (i.e., stretch and shear stress). Here, we describe in detail the use of a custom-developed bioreactor that uniquely enables the decoupling of stretch and shear stress on a tubular scaffold. This allows for the systematic and standardized evaluation of the inflammatory and regenerative capacity of tubular scaffolds under the influence of well-controlled mechanical loads, which we demonstrate on the basis of a dynamic co-culture experiment using human macrophages and myofibroblasts. The key practical steps in this approach&#8212;the construction and setting up of the bioreactor, preparation of the scaffolds and cell seeding, application and maintenance of stretch and shear flow, and sample harvesting for analysis&#8212;are discussed in detail.

This bioreactor was developed to study the individual and combined effects of shear stress and cyclic stretch on vascular tissue growth and remodeling in 3D biomaterial scaffolds. The design of the bioreactor allows for culturing up to eight vascular constructs under various loading conditions (Figure 1A). The vascular constructs are positioned in a flow culture chamber (Figure 1B) in which both the circumferential stretch and WSS can be independently controlled...

Watch this Scientific Journal Video about A Multi-Cue Bioreactor to Evaluate the Inflammatory and Regenerative Capacity of Biomaterials under Flow and Stretch at JoVE.com

A Multi-Cue Bioreactor to Evaluate the Inflammatory and Regenerative Capacity of Biomaterials under Flow and Stretch

The goal of this protocol is to execute a dynamic co-culture of human macrophages and myofibroblasts in tubular electrospun scaffolds to investigate material-driven tissue regeneration, using a bioreactor which enables the decoupling of shear stress and cyclic stretch.

The use of resorbable biomaterials to induce regeneration directly in the body is an attractive strategy from a translational perspective. Such materials ...

a-multi-cue-bioreactor-to-evaluate-inflammatory-regenerative-capacity

Research

JoVE Journal

Engineering

5.5K Views.  Eindhoven University of Technology. Het doel van dit protocol is om een dynamische co-cultuur van menselijke macrofagen en myofibroblasten in buisvormige elektrospunsteigers uit te voeren om materiaalgestuurde weefselregeneratie te onderzoeken, met behulp van een bioreactor die het ontkoppelen van schuifspanning en cyclische rek mogelijk maakt.

Video: A Multi-Cue Bioreactor to Evaluate the Inflammatory and Regenerative Capacity of Biomaterials under Flow and Stretch

Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological environment, both mechanical and chemical stimulation are required to maintain the proper development and functionality of arterial vessels1,2.
Bioreactor culture systems developed by our group are designed to support vessel regeneration within a precisely controlled chemo-mechanical environment mimicking that of native vessels. Our bioreactor assembly and maintenance procedures are fairly simple and highly repeatable3,4. Smooth muscle cells (SMCs) are seeded onto a tubular polyglycolic acid (PGA) mesh that is threaded over compliant silicone tubing and cultured in the bioreactor with or without pulsatile stimulation for up to 12 weeks. There are four main attributes that distinguish our bioreactor from some predecessors. 1) Unlike other culture systems that simulate only the biochemical surrounding of native blood vessels, our bioreactor also creates a physiological pulsatile environment by applying cyclic radial strain to the vessels in culture. 2) Multiple engineered vessels can be cultured simultaneously under different mechanical conditions within a controlled chemical environment. 3) The bioreactor allows a mono layer of endothelial cells (EC) to be easily coated onto the luminal side of engineered vessels for animal implantation models. 4) Our bioreactor can also culture engineered vessels with different diameter size ranged from 1 mm to 3 mm, saving the effort to tailor each individual bioreactor to fit a specific diameter size. 
The engineered vessels cultured in our bioreactor resemble native blood vessels histologically to some degree. Cells in the vessel walls express mature SMC contractile markers such as smooth muscle myosin heavy chain (SMMHC)3. A substantial amount of collagen is deposited within the extracellular matrix, which is responsible for ultimate mechanical strength of the engineered vessels5. Biochemical analysis also indicates that collagen content of engineered vessels is comparable to that of native arteries6. Importantly, the pulsatile bioreactor has consistently regenerated vessels that exhibit mechanical properties that permit successful implantation experiments in animal models3,7. Additionally, this bioreactor can be further modified to allow real-time assessment and tracking of collagen remodeling over time, non-invasively, using a non-linear optical microscopy (NLOM)8. To conclude, this bioreactor should serve as an excellent platform to study the fundamental mechanisms that regulate the regeneration of functional small-diameter vascular grafts.

Our group has developed a bioreactor culture system that mimics the physiological pulsatile stresses of the cardiovascular system to regenerate implantable small-diameter vascular grafts.

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological ...

Bioengineering

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature maintenance and pathological malformations can result in severe ischemic diseases, while overly abundant vascular development is associated with cancer and inflammatory disorders. A promising form of pro-angiogenic therapy is the use of angiogenic cell sources, which can provide regulatory factors as well as physical support for newly developing vasculature.
Mesenchymal Stromal Cells (MSCs) are extensively investigated candidates for vascular regeneration due to their paracrine effects and their ability to detect and home to ischemic or inflamed tissues. In particular, first trimester human umbilical cord perivascular cells (FTM HUCPVCs) are a highly promising candidate due to their pericyte-like properties, high proliferative and multilineage potential, immune-privileged properties, and robust paracrine profile. To effectively evaluate potentially angiogenic regenerative cells, it is a requisite to test them in reliable and &#34;translatable&#34; pre-clinical assays. The aortic ring assay is an ex vivo angiogenesis model that allows for easy quantification of tubular endothelial structures, provides accessory supportive cells and extracellular matrix (ECM) from the host, excludes inflammatory components, and is fast and inexpensive to set up. This is advantageous when compared to in vivo models (e.g., corneal assay, Matrigel plug assay); the aortic ring assay can track the administered cells and observe intercellular interactions while avoiding xeno-immune rejection.
We present a protocol for a novel application of the aortic ring assay, which includes human MSCs in co-cultures with developing rat aortic endothelial networks. This assay allows for the analysis of the MSC contribution to tube formation and development through physical pericyte-like interactions and of their potency for actively migrating to sites of angiogenesis, and for evaluating their ability to perform and mediate ECM processing. This protocol provides further information on changes in MSC phenotype and gene expression following co-culture.

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Here, we present a novel application of the aortic ring assay where prelabelled mesenchymal cells are co-cultured with rat aorta-derived endothelial networks. This novel method allows visualization of Mesenchymal Stromal Cells (MSCs) homing and integration with endothelial networks, quantification of network properties, and evaluation of MSC immunophenotypes and gene expression.

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature ...

Developmental Biology

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need for new solutions for substance testing. Many drugs that have been removed from the market due to drug-induced liver injury released their toxic potential only after several doses of chronic testing in humans. However, a controlled microenvironment is pivotal for long-term multiple dosing experiments,&#160;as even minor alterations in extracellular conditions may greatly influence the cell physiology. We focused within our research program on the generation of a microengineered bioreactor, which can be dynamically perfused by an on-chip pump and combines at least two culture spaces for multi-organ applications. This circulatory system mimics the in vivo conditions of primary cell cultures better and assures a steadier,&#160;more quantifiable extracellular relay of signals to the cells. 
For demonstration purposes, human liver equivalents, generated by aggregating differentiated HepaRG cells with human hepatic stellate cells in hanging drop plates, were cocultured with human skin punch biopsies for up to 28 days inside the microbioreactor. The use of cell culture inserts enables the skin to be cultured at an air-liquid interface, allowing topical substance exposure. The microbioreactor system is capable of supporting these cocultures at near physiologic fluid flow and volume-to-liquid ratios, ensuring stable and organotypic culture conditions. The possibility of long-term cultures enables the repeated exposure to substances. Furthermore, a vascularization of the microfluidic channel circuit using human dermal microvascular endothelial cells yields a physiologically more relevant vascular model.

Here, we present a protocol to coculture primary cells, tissue models and punch biopsies in a microfluidic multi-organ chip for up to 28 days. Human dermal microvascular endothelial cells, liver aggregates and skin biopsies were successfully combined in a common media circulation.

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Biological Compatibility Profile on Biomaterials for Bone Regeneration

Large non-union bone fractures are a significant challenge in orthopedic surgery. Although auto- and allogeneic bone grafts are excellent for healing such lesions, there are potential complications with their use. Thus, material scientists are developing synthetic, biocompatible biomaterials to overcome these problems. In this study, we present a multidisciplinary platform for evaluating biomaterials for bone repair. We combined expertise from bone biology and immunology to develop a platform including in vitro osteoclast (OC) and osteoblast (OB) assays and in vivo mouse models of bone repair, immunogenicity, and allergenicity. We demonstrate how to perform the experiments, summarize the results, and report on biomaterial biocompatibility. In particular, we tested OB viability, differentiation, and mineralization and OC viability and differentiation in the context of &#946;-tricalcium phosphate (&#946;-TCP) disks. We also tested a &#946;-TCP/Collagen (&#946;-TCP/C) foam which is a commercially available material used clinically for bone repair in a critical-sized calvarial bone defect mouse model to determine the effects on the early phase of bone healing. In parallel experiments, we evaluated immune and allergic responses in mice. Our approach generates a biological compatibility profile of a bone biomaterial with a range of parameters necessary for predicting the biocompatibility of biomaterials used for bone healing and repair in patients.

The number of novel biomaterials engineered for repairing large bone lesions is continuously expanding with the aim to enhance bone healing and overcome the complications associated with bone transplantation. Here, we present a multidisciplinary strategy for pre-clinical biocompatibility testing of biomaterials for bone repair.

Large non-union bone fractures are a significant challenge in orthopedic surgery. Although auto- and allogeneic bone grafts are excellent for healing such ...

Designing a Bioreactor to Improve Data Acquisition and Model Throughput of Engineered Cardiac Tissues

Heart failure remains the leading cause of death worldwide, creating a pressing need for better preclinical models of the human heart. Tissue engineering is crucial for basic science cardiac research; in vitro human cell culture eliminates the interspecies differences of animal models, while a more tissue-like 3D environment (e.g., with extracellular matrix and heterocellular coupling) simulates in vivo conditions to a greater extent than traditional two-dimensional culture on plastic Petri dishes. However, each model system requires specialized equipment, for example, custom-designed bioreactors and functional assessment devices. Additionally, these protocols are often complicated, labor-intensive, and plagued by the failure of the small, delicate tissues.
This paper describes a process for generating a robust human engineered cardiac tissue (hECT) model system using induced pluripotent stem-cell-derived cardiomyocytes for the longitudinal measurement of tissue function. Six hECTs with linear strip geometry are cultured in parallel, with each hECT&#160;suspended from a pair of force-sensing polydimethylsiloxane (PDMS) posts attached to PDMS racks. Each post is capped with a black PDMS stable post tracker (SPoT), a new feature that improves the ease of use, throughput, tissue retention, and data quality. The shape allows for the reliable optical tracking of post deflections, yielding improved twitch force tracings with absolute active and passive tension. The cap geometry eliminates tissue failure due to hECTs slipping off the posts, and as they involve a second step after PDMS rack fabrication, the SPoTs can be added to existing PDMS post-based designs without major changes to the bioreactor fabrication process.
The system is used to demonstrate the importance of measuring hECT function at physiological temperatures and shows stable tissue function during data acquisition. In summary, we describe a state-of-the-art model system that reproduces key physiological conditions to advance the biofidelity, efficiency, and rigor of engineered cardiac tissues for in vitro applications.

Three-dimensional cardiac tissues bioengineered using stem-cell-derived cardiomyocytes have emerged as promising models for studying healthy and diseased human myocardium in vitro while recapitulating key aspects of the native cardiac niche. This manuscript describes a protocol for fabricating and analyzing high-content engineered cardiac tissues generated from human induced pluripotent stem-cell-derived cardiomyocytes.

Heart failure remains the leading cause of death worldwide, creating a pressing need for better preclinical models of the human heart. Tissue engineering ...

A Novel 3D Printed Bioreactor for Ex Vivo Culture of Blood Vessels in Perfusion

Ex Vivo Perfusion Culture of Large Blood Vessels in a 3D Printed Bioreactor

Vascular disease forms the basis of most cardiovascular diseases (CVDs), which remain the primary cause of mortality and morbidity worldwide. Efficacious surgical and pharmacological interventions to prevent and treat vascular disease are urgently needed. In part, the shortage of translational models limits the understanding of the cellular and molecular processes involved in vascular disease. Ex vivo perfusion culture bioreactors provide an ideal platform for the study of large animal vessels (including humans) in a controlled dynamic environment, combining the ease of in vitro culture and the complexity of the live tissue. Most bioreactors are, however, custom manufactured and therefore difficult to adopt, limiting the reproducibility of the results. This paper presents a 3D printed system that can be easily produced and applied in any biological lab, and provides a detailed protocol for its setup, enabling users&#39; operation. This innovative and reproducible ex vivo perfusion culture system enables the culture of blood vessels for up to 7 days in physiological conditions. We expect that adopting a standardized perfusion bioreactor will support a better understanding of physiological and pathological processes in large animal blood vessels and accelerate the discovery of new therapeutics.

Author Spotlight: EasyFlow - An Economical and Adaptable Perfusion Bioreactor for Large Blood Vessel Culture 

This protocol presents the setup and operation of a newly developed, 3D printed bioreactor for the ex vivo culture of blood vessels in perfusion. The system is designed to be easily adopted by other users, practical, affordable, and adaptable to different experimental applications, such as basic biology and pharmacological studies.

Vascular disease forms the basis of most cardiovascular diseases (CVDs), which remain the primary cause of mortality and morbidity worldwide. Efficacious ...

A Multi-Cue Bioreactor to Evaluate the Inflammatory and Regenerative Capacity of Biomaterials under Flow and Stretch

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