Channeled Scaffolds Research Articles

3D printed water-soluble scaffolds for rapid production of PDMS micro-fluidic flow chambers

We report a novel method for fabrication of three-dimensional (3D) biocompatible micro-fluidic flow chambers in polydimethylsiloxane (PDMS) by 3D-printing water-soluble polyvinyl alcohol (PVA) filaments as master scaffolds. The scaffolds are first embedded in the PDMS and later residue-free dissolved in water leaving an inscription of the scaffolds in the hardened PDMS. We demonstrate the strength of our method using a regular, cheap 3D printer, and evaluate the inscription process and the channels micro-fluidic properties using image analysis and digital holographic microscopy. Furthermore, we provide a protocol that allows for direct printing on coverslips and we show that flow chambers with a channel cross section down to 40 μm × 300 μm can be realized within 60 min. These flow channels are perfectly transparent, biocompatible and can be used for microscopic applications without further treatment. Our proposed protocols facilitate an easy, fast and adaptable production of micro-fluidic channel designs that are cost-effective, do not require specialized training and can be used for a variety of cell and bacterial assays. To help readers reproduce our micro-fluidic devices, we provide: full preparation protocols, 3D-printing CAD files for channel scaffolds and our custom-made molding device, 3D printer build-plate leveling instructions, and G-code.

Simulation of the effects of oxygen carriers and scaffold geometry on oxygen distribution and cell growth in a channeled scaffold for engineering myocardium

This study proposes a mathematical model to evaluate the impact of oxygen carriers and scaffold geometry on oxygen distribution and cell growth in a 3D cardiac construct using computational fluid dynamics (CFD). Flow equations, oxygen balance equation and cell balance equation were solved using special initial and boundary conditions. The modeling results revealed that 55% increase in cardiac cell density occurred by using 6.4% perfluorocarbon oxygen carrier (PFC) compared to pure culture medium without PFC supplementation. Moreover, the effects of the scaffold geometry on cell density were examined by changing the channel numbers and the construct length. A 30% increase in the average cells density was observed by increasing the channel numbers from 37 to 145. Furthermore, the average cell density was increased 23% by decreasing the scaffold length from 0.5 to 0.2 cm length. Overall, the average cell density of cardiac cells can be increased 2-fold by using PFC oxygen carrier and optimizing the scaffold's geometry, simultaneously.

Xenon for tunnelling analysis of the efflux pump component OprN.

Tripartite efflux pumps are among the main actors responsible for antibiotics resistance in Gram-negative bacteria. In the last two decades, structural studies gave crucial information about the assembly interfaces and the mechanistic motions. Thus rigidifying the assembly seems to be an interesting way to hamper the drug efflux. In this context, xenon is a suitable probe for checking whether small ligands could act as conformational lockers by targeting hydrophobic cavities. Here we focus on OprN, the outer membrane channel of the MexEF efflux pump from Pseudomonas aeruginosa. After exposing OprN crystals to xenon gas pressure, 14 binding sites were observed using X-ray crystallography. These binding sites were unambiguously characterized in hydrophobic cavities of OprN. The major site is observed in the sensitive iris-like region gating the channel at the periplasmic side, built by the three key-residues Leu 405, Asp 109, and Arg 412. This arrangement defines along the tunnel axis a strong hydrophobic/polar gradient able to enhance the passive efflux mechanism of OprN. The other xenon atoms reveal strategic hydrophobic regions of the channel scaffold to target, with the aim to freeze the dynamic movements responsible of the open/close conformational equilibrium in OprN.

Channeled β‐TCP Scaffolds Promoted Vascularization and Bone Augmentation in Mandible of Beagle Dogs

A major hindrance to successful alveolar bone augmentation and ridge preservation using synthetic scaffolds is insufficient vascularization in the implanted bone grafts. The slow ingrowth of host vasculature from the bone bed of alveolar bone to the top of the implanted bone grafts leads to limited bone formation in the upper layers of the implanted grafts, which hinders the subsequent implantation of titanium dental implants. In this study, macroporous beta‐tricalcium phosphate (β‐TCP) scaffolds with multiple vertical hollow channels are fabricated that play a similar role as blood vessels for nutrient diffusion and cell migration. The results show that the hollow channels accelerate the degradation rate of the β‐TCP scaffolds and the in vitro release of a bone forming peptide‐1, which significantly promote proliferation and osteogenesis of human bone mesenchymal stem cells on the channeled scaffolds, compared to nonchanneled scaffolds in vitro. More volume of newly formed bone tissues with more blood vessels are augmented in the channeled scaffolds when implanted in mandibular bone defects of beagle dogs. Channeled scaffolds significantly promote new bone formation and augment the height of the mandible. These findings indicate channeled scaffolds facilitate vascularization and bone formation and have great potential for vascularized bone augmentation.

Role of protein dynamics in ion selectivity and allosteric coupling in the NaK channel

Flux-dependent inactivation that arises from functional coupling between the inner gate and the selectivity filter is widespread in ion channels. The structural basis of this coupling has only been well characterized in KcsA. Here we present NMR data demonstrating structural and dynamic coupling between the selectivity filter and intracellular constriction point in the bacterial nonselective cation channel, NaK. This transmembrane allosteric communication must be structurally different from KcsA because the NaK selectivity filter does not collapse under low-cation conditions. Comparison of NMR spectra of the nonselective NaK and potassium-selective NaK2K indicates that the number of ion binding sites in the selectivity filter shifts the equilibrium distribution of structural states throughout the channel. This finding was unexpected given the nearly identical crystal structure of NaK and NaK2K outside the immediate vicinity of the selectivity filter. Our results highlight the tight structural and dynamic coupling between the selectivity filter and the channel scaffold, which has significant implications for channel function. NaK offers a distinct model to study the physiologically essential connection between ion conduction and channel gating.

In vivo vascularization of anisotropic channeled porous polylactide-based capsules for islet transplantation: the effects of scaffold architecture and implantation site.

The replacement of pancreatic islets for the possible treatment of type 1 diabetes is limited by the extremely high oxygen demand of the islets. To this end, here we hypothesize to create a novel extra-hepatic highly-vascularized bioartificial cavity using a porous scaffold as a template and using the host body as a living bioreactor for subsequent islet transplantation. Polylactide-based capsular-shaped anisotropic channeled porous scaffolds were prepared by following the unidirectional thermally-induced phase separation technique, and were implanted under the skin and in the greater omentum of Brown Norway rats. Polyamide mesh-based isotropic regular porous capsules were used as the controls. After 4weeks, the implants were excised and analyzed by histology. The hematoxylin and eosin, as well as Masson's trichrome staining, revealed a) low or no infiltration of giant inflammatory cells in the implant, b) minor but insignificant fibrosis around the implant, c) guided infiltration of host cells in the test capsule in contrast to random cell infiltration in the control capsule, and d) relatively superior cell infiltration in the capsules implanted in the greater omentum than in the capsules implanted under the skin. Furthermore, the anti-CD31 immunohistochemistry staining revealed numerous vessels at the implant site, but mostly on the external surface of the capsules. Taken together, the current study, the first of its kind, is a significant step-forward towards engineering a bioartificial microenvironment for the transplantation of islets.

Channeled polymeric scaffolds with polypeptide gel filling for lengthwise guidance of neural cells

CNS damages are often irreversible since neurons of the central nervous system are unable to regenerate after an injury. As a new strategy within the nervous system tissue engineering, multifunctional systems based on two different biomaterials to support axonal guidance in damaged connective tracts have been developed herein. These systems are composed of a channeled scaffold made of ethyl acrylate and hydroxyethyl acrylate copolymer, P(EA-co-HEA), with parallel tubular micropores, combined with an injectable and in situ gelable self-assembling polypeptide (RAD16-I) as pores filler. The polymer scaffold is intended to provide a three-dimensional context for axon growth; subsequently, its morphology and physicochemical parameters have been determined by scanning electron microscopy, density measurements and compression tests. Besides, the hydrogel acts as a cell-friendly nanoenvironment while it creates a gradient of bioactive molecules (nerve growth factor, NGF) along the scaffolds channels; the chemotactic effect of NGF has been evaluated by a quantitative ELISA assay. These multifunctional systems have shown ability to keep circulating NGF, as well as proper short-term in vitro biological response with glial cells and neural progenitors.

Neural tissue regeneration in experimental brain injury model with channeled scaffolds of acrylate copolymers

The objective of the present study was to evaluate the biocompatibility and cell hosting ability of a copolymer scaffold based on ethyl acrylate (EA) and hydroxyl ethyl acrylate (HEA) in vivo after an experimental brain injury. Wistar rats were subjected to cryogenic traumatic brain injury. We evaluated the tissue response to the implanted materials after 8 weeks. The materials were implanted devoid of cells; they provoked a minimal scar response by the host tissue and permitted the invasion of neurons and glia inside them. We also found new blood vessels surrounding and inside the implant. Thus, the copolymer scaffold proves to offer a suitable environment producing a cellular network potentially useful in brain repair after brain injury.

Vascularization of hollow channel-modified porous silk scaffolds with endothelial cells for tissue regeneration

Despite the promise for stem cell-based tissue engineering for regenerative therapy, slow and insufficient vascularization of large tissue constructs negatively impacts the survival and function of these transplanted cells. A combination of channeled porous silk scaffolds and prevascularization with endothelial cells was investigated to test the ability of this tissue engineering strategy to support rapid and extensive vascularization process. We report that hollow channels promote in vitro prevascularization by facilitating endothelial cell growth, VEGF secretion, and capillary-like tube formation. When implanted in vivo, the pre-established vascular networks in the hollow channel scaffolds anastomose with host vessels and exhibit accelerated vascular infiltration throughout the whole tissue construct, which provides timely and sufficient nutrients to ensure the survival of the transplanted stem cells. This tissue engineering strategy can promote the effective application of stem cell-based regeneration to improve future clinical applications.

Engineering a vascularized collagen-β-tricalcium phosphate graft using an electrochemical approach

Vascularization of three-dimensional large synthetic grafts for tissue regeneration remains a significant challenge. Here we demonstrate an electrochemical approach, named the cell electrochemical detachment (CED) technique, to form an integral endothelium and use it to prevascularize a collagen-β-tricalcium phosphate (β-TCP) graft. The CED technique electrochemically detached an integral endothelium from a gold-coated glass rod to a collagen-infiltrated, channeled, macroporous β-TCP scaffold, forming an endothelium-lined microchannel containing graft upon removal of the rod. The in vitro results from static and perfusion culture showed that the endothelium robustly emanated microvascular sprouting and prevascularized the entire collagen/β-TCP integrated graft. The in vivo subcutaneous implantation studies showed that the prevascularized collagen/β-TCP grafts established blood flow originating from the endothelium-lined microchannel within a week, and the blood flow covered more areas in the graft over time. In addition, many blood vessels invaded the prevascularized collagen/β-TCP graft and the in vitro preformed microvascular networks anastomosed with the host vasculature, while collagen alone without the support of rigid ceramic scaffold showed less blood vessel invasion and anastomosis. These results suggest a promising strategy for effectively vascularizing large tissue-engineered grafts by integrating multiple hydrogel-based CED-engineered endothelium-lined microchannels into a rigid channeled macroporous scaffold.

Microfabrication of channel arrays promotes vessel-like network formation in cardiac cell construct and vascularization in vivo

Pre-vascularization is important for the reconstruction of dense and metabolically active myocardial tissue and its integration with the host myocardium after implantation. Herein, we demonstrate that the fabrication of micro-channels in alginate scaffold combined with the presentation of adhesion peptides and an angiogenic growth factor promote vessel-like networks in the construct, both in vitro and in vivo. Using a CO2 laser engraving system, 200 µm diameter channels were formed from top to bottom of the 2 mm thick alginate scaffold, with a channel-to-channel distance of 400 µm. Cells were seeded in a sequential manner onto the scaffolds: first, human umbilical vascular endothelial cells (HUVECs) were seeded and cultured for three days, then neonatal rat cardiomyocytes (CMs) and cardiofibroblasts were added at a final cell ratio of 50:35:15, respectively, and the constructs were cultivated for an additional seven days. A vessel-like network was formed within the cell constructs, wherein HUVECs were organized around the channels in a multilayer manner, while the CMs were located in-between the channels and exhibited the characteristic morphological features of a mature cardiac fiber. Acellular scaffolds with the affinity-bound basic fibroblast growth factor were implanted subcutaneously in mice. Increased cell penetration into the channeled scaffold and greater vessel density were found in comparison with the nonchanneled scaffolds. Our results thus point to the importance of micro-channels as a major structural promoter of vascularization in scaffolds, in conjunction with the sequential preculture of ECs and angiogenic factor presentation.

The effect of controlled expression of VEGF by transduced myoblasts in a cardiac patch on vascularization in a mouse model of myocardial infarction

Key requirements for cardiac tissue engineering include the maintenance of cell viability and function and the establishment of a perfusable vascular network in millimeters thick and compact cardiac constructs upon implantation. We investigated if these requirements can be met by providing an intrinsic vascularization stimulus (via sustained action of VEGF secreted at a controlled rate by transduced myoblasts) to a cardiac patch engineered under conditions of effective oxygen supply (via medium flow through channeled elastomeric scaffolds seeded with neonatal cardiomyocytes). We demonstrate that this combined approach resulted in increased viability, vascularization and functionality of the cardiac patch. After implantation in a mouse model of myocardial infarction, VEGF-expressing patches displayed significantly improved engraftment, survival and differentiation of cardiomyocytes, leading to greatly enhanced contractility as compared to controls not expressing VEGF. Controlled VEGF expression also mediated the formation of mature vascular networks, both within the engineered patches and in the underlying ischemic myocardium. We propose that this combined cell-biomaterial approach can be a promising strategy to engineer cardiac patches with intrinsic and extrinsic vascularization potential.

Fabrication of multilayer ZrO2–biphasic calcium phosphate–poly-caprolactone unidirectional channeled scaffold for bone tissue formation

We developed a continuously porous scaffold with laminated matrix and bone-like microstructure by a multi-pass extrusion process. In this scaffold, tetragonal ZrO₂, biphasic calcium phosphate and poly-caprolactone layers were arranged in a co-axially laminated unit cell with a channel in the center. The entire matrix phase had a laminated microstructure of alternate lamina of tetragonal ZrO₂, biphasic calcium phosphate and poly-caprolactone--biphasic calcium phosphate with optimized designed thickness and channeled porosity. Each of the continuous pores was coaxially encircled by the poly-caprolactone--biphasic calcium phosphate layer, biphasic calcium phosphate layer and finally tetragonal ZrO₂ layer, one after the other. Before extrusion, 5 vol% graphite powder was mixed with tetragonal ZrO₂ to ensure pores in the outer layer and connectivity among the lamellas. The design strategy is aimed to incorporate a lamellar microstructure like the natural bone in the macro-scaled ceramic body to investigate the strengthening phenomenon and pave the way for fabricating complex microstructure of natural bone could be applied for whole bone replacement. The final fabricated scaffold had a compressive strength of 12.7 MPa and porosity of 78 vol% with excellent cell viability, cell attachment and osteocalcin and collagen expression from cultured MG63 cells on scaffold.

Fabrication of channeled scaffolds with ordered array of micro-pores through microsphere leaching and indirect Rapid Prototyping technique

Advanced scaffold fabrication techniques such as Rapid Prototyping (RP) are generally recognized to be advantageous over conventional fabrication methods in terms architectural control and reproducibility. Yet, most RP techniques tend to suffer from resolution limitations which result in scaffolds with uncontrollable, random-size pores and low porosity, albeit having interconnected channels which is characteristically present in most RP scaffolds. With the increasing number of studies demonstrating the profound influences of scaffold pore architecture on cell behavior and overall tissue growth, a scaffold fabrication method with sufficient architectural control becomes imperative. The present study demonstrates the use of RP fabrication techniques to create scaffolds having interconnected channels as well as controllable micro-size pores. Adopted from the concepts of porogen leaching and indirect RP techniques, the proposed fabrication method uses monodisperse microspheres to create an ordered, hexagonal closed packed (HCP) array of micro-pores that surrounds the existing channels of the RP scaffold. The pore structure of the scaffold is shaped using a single sacrificial construct which comprises the microspheres and a dissolvable RP mold that were sintered together. As such, the size of pores as well as the channel configuration of the scaffold can be tailored based on the design of the RP mold and the size of microspheres used. The fabrication method developed in this work can be a promising alternative way of preparing scaffolds with customized pore structures that may be required for specific studies concerning cell-scaffold interactions.

Microfabrication of cylindrical microfluidic channel networks for microvascular research

Current methods for formation of microvascular channel scaffolds are limited with non-circular channel cross-sections, complicated fabrication, and less flexibility in microchannel network design. To address current limitations in the creation of engineered microvascular channels with complex three-dimensional (3-D) geometries in the shape of microvessels, we have developed a reproducible, cost-effective, and flexible micromanufacturing process combined with photolithographic reflowable photoresist and soft lithography techniques to fabricate cylindrical microchannel and networks. A positive reflowable photoresist AZ P4620 was used to fabricate a master microchannel mold with semi-circular cross-sections. By the alignment and bonding of two polydimethylsiloxane (PDMS) microchannels replicated from the master mold together, a cylindrical microchannel or microchannel network was created. Further examination of the channel dimensions and surface profiles at different branching levels showed that the shape of the microfluidic channel was well approximated by a semi-circular surface, and a multi-level, multi-depth channel network was created. In addition, a computational fluidic dynamics (CFD) model was used to simulate shear flows and corresponding pressure distributions inside of the microchannel and channel network based on the dimensions of the fabricated channels. The fabricated multi-depth cylindrical microchannel network can provide platforms for the investigation of microvascular cells growing inside of cylindrical channels under shear flows and lumen pressures, and work as scaffolds for the investigation of morphogenesis and tubulogenesis.

Development of channeled nanofibrous scaffolds for oriented tissue engineering.

A tissue-engineering scaffold resembling the structure of the natural extracellular matrix can often facilitate tissue regeneration. Nerve and tendon are oriented micro-scale tissue bundles. In this study, a method combining injection molding and thermally induced phase separation techniques is developed to create single- and multiple-channeled nanofibrous poly(L-lactic acid) scaffolds. The overall shape, the number and spatial arrangement of channels, the channel wall matrix architecture, the porosity and mechanical properties of the scaffolds are all tunable. The porous NF channel wall matrix provides an excellent microenvironment for protein adsorption and the attachment of PC12 neuronal cells and tendon fibroblast cells, showing potential for neural and tendon tissue regeneration.

Engineered channels enhance cellular density in perfused scaffolds

Scaffold-based tissue engineering provides cells with an engineered matrix to enhance and direct cell attachment, proliferation and differentiation. One critical limitation to current tissue engineering approaches is the inability to create densely populated constructs thicker than a few 100 μm. We hypothesized that development of porous, channeled scaffolds would increase cell density and uniformity of their spatial distribution through scaffold channel perfusion. Patterned polyurethane sheets were fabricated using a sprayed phase separation technique and laminated together to form 1.5 mm thick channeled scaffolds. Hydraulic permeability testing confirmed the presence of functional channels throughout the multilaminate construct. A continuous flow bioreactor was used to perfuse the construct with medium during the culture period. Cross-sectional cell densities and spatial uniformities were measured in channeled and nonchanneled scaffolds under different seeding and culture conditions. Channeled scaffolds were found to have higher densities of human mesenchymal stem cells than nonchanneled samples. Perfused scaffolds had more uniform spatial distribution of cells within the scaffold compared to statically cultured scaffolds. In conclusion, we have shown the channeled scaffolds to be a promising approach toward creating thick tissue-engineered constructs.

Sustained Delivery of Dibutyryl Cyclic Adenosine Monophosphate to the Transected Spinal Cord Via Oligo [(Polyethylene Glycol) Fumarate] Hydrogels

This study describes the use of oligo [(polyethylene glycol) fumarate] (OPF) hydrogel scaffolds as vehicles for sustained delivery of dibutyryl cyclic adenosine monophosphate (dbcAMP) to the transected spinal cord. dbcAMP was encapsulated in poly(lactic-co-glycolic acid) (PLGA) microspheres, which were embedded within the scaffolds architecture. Functionality of the released dbcAMP was assessed using neurite outgrowth assays in PC12 cells and by delivery to the transected spinal cord within OPF seven channel scaffolds, which had been loaded with Schwann cells or mesenchymal stem cells (MSCs). Our results showed that encapsulation of dbcAMP in microspheres lead to prolonged release and continued functionality in vitro. These microspheres were then successfully incorporated into OPF scaffolds and implanted in the transected thoracic spinal cord. Sustained delivery of dbcAMP inhibited axonal regeneration in the presence of Schwann cells but rescued MSC-induced inhibition of axonal regeneration. dbcAMP was also shown to reduce capillary formation in the presence of MSCs, which was coupled with significant functional improvements. Our findings demonstrate the feasibility of incorporating PLGA microsphere technology for spinal cord transection studies. It represents a novel sustained delivery mechanism within the transected spinal cord and provides a platform for potential delivery of other therapeutic agents.

Novel use of X-ray micro computed tomography to image rat sciatic nerve and integration into scaffold

This paper describes how specimens of nervous tissue can be prepared for successful imaging in X-ray Micro Computed Tomography (μCT), and how this method can be used to study the integration of nervous tissue into a polymeric scaffold. The sample preparation involves staining the biological tissue with osmium tetroxide to increase its X-ray attenuation, and a technique for maintaining the specimen in a moist environment during the experiment to prevent drying and shrinkage. Using this method it was possible to observe individual nerve fascicles and their relationship to the 3-D tissue structure. A scaffold supporting a regenerated sciatic nerve was similarly stained to distinguish the nervous tissue from the scaffold, and to observe how the nerve grew through a 2.5mm long, 100μm×100μm cross-section channel polyimide array. Furthermore, blood vessels could be identified in these images, and it was possible to monitor how a large proximal blood vessel split through the channel scaffold and proceeded down individual channels. This paper explains how μCT is a useful tool both for studying the location and extent of growth into a polymeric scaffold, and for determining whether the regenerated tissue has blood supply.

Perfusion seeding of channeled elastomeric scaffolds with myocytes and endothelial cells for cardiac tissue engineering

The requirements for engineering clinically sized cardiac constructs include medium perfusion (to maintain cell viability throughout the construct volume) and the protection of cardiac myocytes from hydrodynamic shear. To reconcile these conflicting requirements, we proposed the use of porous elastomeric scaffolds with an array of channels providing conduits for medium perfusion, and sized to provide efficient transport of oxygen to the cells, by a combination of convective flow and molecular diffusion over short distances between the channels. In this study, we investigate the conditions for perfusion seeding of channeled constructs with myocytes and endothelial cells without the gel carrier we previously used to lock the cells within the scaffold pores. We first established the flow parameters for perfusion seeding of porous elastomer scaffolds using the C2C12 myoblast line, and determined that a linear perfusion velocity of 1.0 mm/s resulted in seeding efficiency of 87% +/- 26% within 2 hours. When applied to seeding of channeled scaffolds with neonatal rat cardiac myocytes, these conditions also resulted in high efficiency (77.2% +/- 23.7%) of cell seeding. Uniform spatial cell distributions were obtained when scaffolds were stacked on top of one another in perfusion cartridges, effectively closing off the channels during perfusion seeding. Perfusion seeding of single scaffolds resulted in preferential cell attachment at the channel surfaces, and was employed for seeding scaffolds with rat aortic endothelial cells. We thus propose that these techniques can be utilized to engineer thick and compact cardiac constructs with parallel channels lined with endothelial cells.