Channeled Scaffolds Research Articles

Branched Channels in Porous β-Tricalcium Phosphate Scaffold Promote Vascularization.

Rapid and efficient vascularization is still considerably challenging for a porous β-tricalcium phosphate (β-TCP) scaffold to achieve. To overcome this challenge, branched channels were created in the porous β-TCP scaffold by using 3D printing and a template-casting method to facilitate the instant flow of blood supply. Human bone mesenchymal stem cells (hBMSCs) and human umbilical vein endothelial cells (HUVECs) were seeded in the channeled porous scaffolds and characterized through a double-stranded DNA (dsDNA) assay, alkaline phosphatase (ALP) assay, and cell migration. Channeled porous β-TCP scaffolds were then implanted in the subcutaneous pockets of mice. Histological staining and immunohistochemical staining on vascularization and bone-related markers were carried out on the embedded paraffin sections. Results from in vitro experiments showed that branched channels significantly promoted HUVECs' infiltration, migration, proliferation, and angiogenesis, and also promoted the proliferation and osteogenesis differentiation of hBMSCs. In vivo implantation results showed that, in the early stage after implantation, cells significantly migrated into branched channeled scaffolds. More matured blood vessels formed in the branched channeled scaffolds compared to that in nonchanneled and straight channeled scaffolds. Beside promoting vascularization, the branched channels also stimulated the infiltration of bone-related cells into the scaffolds. These results suggested that the geometric design of branched channels in the porous β-TCP scaffold promoted rapid vascularization and potentially stimulated bone cells recruitment.

Design and optimization of 3D-bioprinted cell-laden scaffolds in dynamic culture

Light-based 3D printing enables the fabrication of biological scaffolds with high precision, versatility and biocompatibility, particularly the cell-laden scaffolds with architecturally complex geometric features. However, many bioprinted tissue scaffolds suffer from low cell viability due to insufficient oxygen and nutrient supply, which is heavily influenced by scaffold structure and cultivation conditions. Current practice relies mainly on resource-intensive trial-and-error methods to optimize scaffolds’ structures and cultivation parameters. In this study, we developed a comprehensive multi-physics model integrating fluid dynamics, oxygen mass transfer, cell oxygen consumption, and cell growth processes to capture cell growth behaviors in scaffolds, establishing a robust theoretical foundation for scaffold structure optimization. The modeling results showed that a large number of parameters, such as system inlet flow rate, geometric feature size, cell parameters, and material properties, significantly impact oxygen concentration and cell growth within the scaffold. A two-step optimization strategy is proposed in this paper and was applied to obtain optimal geometric parameters of channeled scaffolds to demonstrate the model’s effectiveness for scaffold optimization. The model can be employed for scaffolds with arbitrary shapes and various materials, facilitating the optimal design of sophisticated scaffolds for more advanced tissue engineering.

Overlap in synaptic neurological condition susceptibility pathways and the neural pannexin 1 interactome revealed by bioinformatics analyses

ABSTRACT Many neurological conditions exhibit synaptic impairments, suggesting mechanistic convergence. Additionally, the pannexin 1 (PANX1) channel and signaling scaffold is linked to several of these neurological conditions and is an emerging regulator of synaptic development and plasticity; however, its synaptic pathogenic contributions are relatively unexplored. To this end, we explored connections between synaptic neurodevelopmental disorder and neurodegenerative disease susceptibility genes discovered by genome-wide association studies (GWASs), and the neural PANX1 interactome (483 proteins) identified from mouse Neuro2a (N2a) cells. To identify shared susceptibility genes, we compared synaptic suggestive GWAS candidate genes amongst autism spectrum disorders, schizophrenia, Parkinson’s disease, and Alzheimer’s disease. To further probe PANX1 signaling pathways at the synapse, we used bioinformatics tools to identify PANX1 interactome signaling pathways and protein–protein interaction clusters. To shed light on synaptic disease mechanisms potentially linking PANX1 and these four neurological conditions, we performed additional cross-analyses between gene ontologies enriched for the PANX1 synaptic and disease-susceptibility gene sets. Finally, to explore the regional specificity of synaptic PANX1-neurological condition connections, we identified brain region-specific elevations of synaptic PANX1 interactome and GWAS candidate gene set transcripts. Our results confirm considerable overlap in risk genes for autism spectrum disorders and schizophrenia and identify potential commonalities in genetic susceptibility for neurodevelopmental disorders and neurodegenerative diseases. Our findings also pinpointed novel putative PANX1 links to synaptic disease-associated pathways, such as regulation of vesicular trafficking and proteostasis, warranting further validation.

3D printed hollow channeled hydrogel scaffolds with antibacterial and wound healing activities

The development of hydrogel based scaffold with the capability of enhanced antibacterial effects and wound healing is the promising strategy for the treatment of wound tissues with bacterial infection. Herein, we fabricated a hollow channeled hydrogel scaffold based on the mixture of dopamine modified alginate (Alg-DA) and gelatin via co-axial 3D printing for the treatment of bacterial-infected wound. The scaffold was crosslinked by copper/calcium ions, which could enhance the structural stability and mechanical properties. Meanwhile, copper ions crosslinking endowed the scaffold with good photothermal effects. The photothermal effect and copper ions showed excellent antibacterial activity against both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria. Moreover, the hollow channels and the sustained released copper ions could stimulate angiogenesis and accelerate wound healing process. Thus, the prepared hollow channeled hydrogel scaffold might be a potential candidate for promoting wound healing application.

Design of IKVAV peptide/gold nanoparticle decorated, micro/nano-channeled PCL/PLGA film scaffolds for neuronal differentiation and neurite outgrowth

In the field of neural tissue engineering, intensive efforts are being made to develop tissue scaffolds that can support an effective functional recovery and neural development by guiding damaged axons and neurites. Micro/nano-channeled conductive biomaterials are considered a promising approach for repairing the injured neural tissues. Many studies have demonstrated that the micro/nano-channels and aligned nanofibers could guide the neurites to extend along the direction of alignment. However, an ideal biocompatible scaffold containing conductive arrays that could promote effective neural stem cell differentiation and development, and also stimulate high neurite guidance has not been fully developed. In the current study, we aimed to fabricate micro/nano-channeled polycaprolactone (PCL)/Poly-d,l-lactic-co-glycolic acid (PLGA) hybrid film scaffolds, decorate their surfaces with IKVAV pentapeptide/gold nanoparticles (AuNPs), and investigate the behavior of PC12 cells and neural stem cells (NSCs) on the developed biomaterial under static/bioreactor conditions. Here we show that channeled groups decorated with AuNPs highly promote neurite outgrowth and neuronal differentiation along linear lines in the presence of electrical stimulation, compared with the polypyrrole (PPy) coating, which has been used traditionally for many years. Hopefully, this newly developed channeled scaffold structure (PCL/PLGA-AuNPs-IKVAV) could help to support long-distance axonal regeneration and neuronal development after different neural damages.

Flow channel performance in 3D printed hydroxyapatite scaffolds to improve metabolism and tissue ingrowth in flat bone repair

The regeneration of flat bone present great challenges owing to the unique metabolic orientation and the great demand for nutrient transport. Reasonable structural design can effectively improve the performance of tissue regeneration of the scaffolds. In this study, bioactive hydroxyapatite scaffolds with various flow channel designs were precisely produced by 3D printing. The mechanical properties, permeability, substance transport and tissue ingrowth capacity brought by flow channel were investigated. Theoretical simulations and experimental results showed that the mechanical properties were highly correlated with the porosity of the scaffolds rather than the flow channel configurations. Curved channel showed a higher permeability for its better ability of omnidirectional diffusion, enhancing the substance transport and cell settlement by forming vortex flow. The flow channel scaffolds had better tissue ingrowth capability due to the abundant blood supply and cells/nutrients enrichment. Especially, the curved flow channel coupling with the annular one showed the best comprehensive performance in all the configurations designed, leading to the efficiency improvement of tissue ingrowth and bone regeneration. This study proposed preliminary principles for the design of planar two-dimensional flow channel based on the characteristics of flat bone, which provided a potential feasible strategy for large-scale flat bone regeneration and repair.

3D geometry orchestrates the transcriptional landscape of metastatic neuroblastoma cells in a multicellular in vitro bone model.

A key challenge for the discovery of novel molecular targets and therapeutics against pediatric bone metastatic disease is the lack of bona fide in vitro cell models. Here, we show that a beta-tricalcium phosphate (β-TCP) multicellular 3D in vitro bone microtissue model reconstitutes key phenotypic and transcriptional patterns of native metastatic tumor cells while promoting their stemness and proinvasive features. Comparing planar with interconnected channeled scaffolds, we identified geometry as a dominant orchestrator of proangiogenic traits in neuroblastoma tumor cells. On the other hand, the β-TCP-determined gene signature was DNA replication related. Jointly, the geometry and chemical impact of β-TCP revealed a prometastatic landscape of the engineered tumor microenvironment. The proposed 3D multicellular in vitro model of pediatric bone metastatic disease may advance further analysis of the molecular, genetic and metabolic bases of the disease and allow more efficient preclinical target validations.

Artificial Water Channels—Progress Innovations and Prospects

Artificial Water Channels—Progress Innovations and Prospects

Structure-guided unlocking of NaX reveals a non-selective tetrodotoxin-sensitive cation channel

Unlike classical voltage-gated sodium (NaV) channels, NaX has been characterized as a voltage-insensitive, tetrodotoxin-resistant, sodium (Na+)-activated channel involved in regulating Na+ homeostasis. However, NaX remains refractory to functional characterization in traditional heterologous systems. Here, to gain insight into its atypical physiology, we determine structures of the human NaX channel in complex with the auxiliary β3-subunit. NaX reveals structural alterations within the selectivity filter, voltage sensor-like domains, and pore module. We do not identify an extracellular Na+-sensor or any evidence for a Na+-based activation mechanism in NaX. Instead, the S6-gate remains closed, membrane lipids fill the central cavity, and the domain III-IV linker restricts S6-dilation. We use protein engineering to identify three pore-wetting mutations targeting the hydrophobic S6-gate that unlock a robust voltage-insensitive leak conductance. This constitutively active NaX-QTT channel construct is non-selective among monovalent cations, inhibited by extracellular calcium, and sensitive to classical NaV channel blockers, including tetrodotoxin. Our findings highlight a functional diversity across the NaV channel scaffold, reshape our understanding of NaX physiology, and provide a template to demystify recalcitrant ion channels.

Elimination of Induced Hypoxic Regions in Depth of 3D Porous Silk Scaffolds by the Introduction of Channel Configuration.

Development of large, clinically sized tissue constructs with efficient mass transport is a tremendous need in tissue engineering. One major challenge in large tissue-engineered constructs is to support homogeneous delivery of oxygen and nutrients throughout the tissue scaffold while eliminating induced hypoxic regions in depth. To address this goal, we introduced an especial channeled architecture on porous silk-based tissue scaffolds to improve supplying of oxygen to the cells in central regions of the scaffolds. Oxygen gradients were measured and evaluated in three scaffold prototypes, namely, one unchanneled and two channeled scaffolds with different channel diameters (500 μm and 1000 μm). The channels were introduced into the constructs using stainless-steel rods arranged uniformly in stainless-steel mold, a fabrication method that enables precise control over channel diameter and the distance between channels. During 2-week culture of G292 cells, the 1000 μm channeled scaffolds demonstrated higher oxygen concentration at the center compared to 500 μm channeled prototype; however, the oxygen concentration approached the same level around the last days of culture. Nevertheless, homogenous oxygen distribution throughout the 1000 μm channeled constructs and the consequence of higher cell proliferation at day 14 postseeding corroborate the efficient elimination of induced hypoxic regions; and therefore, it holds promise for clinically relevant sized scaffold especially in bone tissue engineering.

Fabrication of channeled scaffolds through polyelectrolyte complex (PEC) printed sacrificial templates for tissue formation.

One of the pivotal factors that limit the clinical translation of tissue engineering is the inability to create large volume and complex three-dimensional (3D) tissues, mainly due to the lack of long-range mass transport with many current scaffolds. Here we present a simple yet robust sacrificial strategy to create hierarchical and perfusable microchannel networks within versatile scaffolds via the combination of embedded 3D printing (EB3DP), tunable polyelectrolyte complexes (PEC), and casting methods. The sacrificial templates of PEC filaments (diameter from 120 to 500 μm) with arbitrary 3D configurations were fabricated by EB3DP and then incorporated into various castable matrices (e.g., hydrogels, organic solutions, meltable polymers, etc.). Rapid dissolution of PEC templates within a 2.00 M potassium bromide aqueous solution led to the high fidelity formation of interconnected channels for free mass exchange. The efficacy of such channeled scaffolds for in vitro tissue formation was demonstrated with mouse fibroblasts, showing continuous cell proliferation and ECM deposition. Subcutaneous implantation of channeled silk fibroin (SF) scaffolds with a porosity of 76% could lead to tissue ingrowth as high as 53% in contrast to 5% for those non-channeled controls after 4 weeks. Both histological and immunofluorescence analyses demonstrated that such channeled scaffolds promoted cellularization, vascularization, and host integration along with immunoregulation.

Microfluidic 3D Printing Responsive Scaffolds with Biomimetic Enrichment Channels for Bone Regeneration

AbstractTissue‐engineered scaffolds have been extensively explored for treating bone defects; however, slow and insufficient vascularization throughout the scaffolds remains a key challenge for further application. Herein, a versatile microfluidic 3D printing strategy to fabricate black phosphorus (BP) incorporated fibrous scaffolds with photothermal responsive channels for improving vascularization and bone regeneration is proposed. The thermal channeled scaffolds display reversible shrinkage and swelling behavior controlled by near‐infrared irradiation, which facilitates the penetration of suspended cells into the scaffold channels and promotes the prevascularization. Furthermore, the embedded BP nanosheets exhibit intrinsic properties for in situ biomineralization and improve in vitro cell proliferation and osteogenic differentiation. Following transplantation in vivo, these channels also promote host vessel infiltration deep into the scaffolds and effectively accelerate the healing process of bone defects. Thus, it is believed that these near‐infrared responsive channeled scaffolds are promising candidates for tissue/vascular ingrowth in diverse tissue engineering applications.

Combining a Vascular Bundle and 3D Printed Scaffold with BMP-2 Improves Bone Repair and Angiogenesis.

Vascularization is currently considered the biggest challenge in bone tissue engineering due to necrosis in the center of large scaffolds. We established a new expendable vascular bundle model to vascularize a three-dimensional printed channeled scaffold with and without bone morphogenetic protein-2 (BMP-2) for improved healing of large segmental bone defects. Bone formation and angiogenesis in an 8 mm critical-sized bone defect in the rat femur were significantly promoted by inserting a bundle consisting of the superficial epigastric artery and vein into the central channel of a large porous polycaprolactone scaffold. Vessels were observed sprouting from the vascular bundle inserted in the central tunnel. Although the regenerated bone volume in the group receiving the scaffold and vascular bundle was similar to that of the healthy femur, the rate of union of the group was not satisfactory (25% at 8 weeks). BMP-2 delivery was found to promote not only bone formation but also angiogenesis in the critical-sized bone defects. Both insertion of the vascular bundle alone and BMP-2 loading alone induced comparable levels of angiogenesis and when used in combination, significantly greater vascular volume was observed. These findings suggest a promising new modality of treatment in large bone defects. Level of Evidence: Therapeutic level I. Impact statement Vascularization is currently the main challenge in bone tissue engineering. The combination of a vascular bundle and an osteoinductive three-dimensional printed graft significantly improved and accelerated bone regeneration and angiogenesis in critical-sized large bone defects, suggesting a promising new modality of treatment in large bone defects.

Effects of Macro-/Micro-Channels on Vascularization and Immune Response of Tissue Engineering Scaffolds.

Although the use of porous scaffolds in tissue engineering has been relatively successful, there are still many limitations that need to be addressed, such as low vascularization, low oxygen and nutrient levels, and immune-induced inflammation. As a result, the current porous scaffolds are insufficient when treating large defects. This paper analyzed scientific research pertaining to the effects of macro-/micro-channels on the cell recruitment, vascularization, and immune response of tissue engineering scaffolds. Most of the studies contained either cell culturing experimentation or experimentation on small animals such as rats and mice. The sacrificial template method, template casting method, and 3D printing method were the most common methods in the fabrication of channeled scaffolds. Some studies combine the sacrificial and 3D printing methods to design and create their scaffold with channels. The overall results from these studies showed that the incorporation of channels within scaffolds greatly increased vascularization, reduced immune response, and was much more beneficial for cell and growth factor recruitment compared with control groups that contained no channels. More research on the effect of micro-/macro-channels on vascularization or immune response in animal models is necessary in the future in order to achieve clinical translation.

Anion Transmembrane Nanochannels from Pore-Forming Helices Constructed by the Dynamic Covalent Reaction of Dihydrazide and Dialdehyde Units.

Anion transmembrane nanochannels constructed from dynamic covalent helices (DCHs) are reported. The dynamic covalent structures can be synthesized by one-pot dynamic covalent reactions and helically self-fold into nanotubes through intramolecular hydrogen bonding and π-π interactions. Such helical structures can vertically self-assemble into long nanofibers under π-π stacking and their hollow nanocavities finally form ion permeation pathways across the lipid membranes. Single-channel electrophysiology signals provide solid evidence of DCHs following the channel rather than the carrier mechanism. Owing to the pore-forming capacity of DCHs, nanochannels are able to accelerate the movement of anions across lipid membranes with high transport activity (EC50 =0.08 mol %). Moreover, DCH channels show dehydration energy dependent anion selectivity. This report highlights the importance of such DCHs as general channel scaffolds with economical synthesis and special nanocavities.

Ligand Induced CuII Transport Restricts Cancer and Mycobacterial Growth: Towards a Plug-and-Select Ion Channel Scaffold.

Synthetic channels with high ion selectivity are attractive drug targets for diseases involving ion dysregulation. Achieving selective transport of divalent ions is highly challenging due their high hydration energies. A small tripeptide amphiphilic scaffold installed with a pybox ligand selectively transports CuII ions across membranes. The peptide forms stable dimeric pores in the membrane and transports ions by a Cu2+ /H+ antiport mechanism. The ligand-induced excellent CuII selectivity as well as high membrane permeability of the peptide is exploited to promote cancer cell death. The peptide's ability to restrict mycobacterial growth serves as seeds to evolve antibacterial strategies centred on selectively modulating ion homeostasis in pathogens. This simple peptide can potentially function as a universal, yet versatile, scaffold wherein the ion selectivity can be precisely controlled by modifying the ligand at the C terminus.

Negligible-cost microfluidic device fabrication using 3D-printed interconnecting channel scaffolds.

This paper reports a novel, negligible-cost and open-source process for the rapid prototyping of complex microfluidic devices in polydimethylsiloxane (PDMS) using 3D-printed interconnecting microchannel scaffolds. These single-extrusion scaffolds are designed with interconnecting ends and used to quickly configure complex microfluidic systems before being embedded in PDMS to produce an imprint of the microfluidic configuration. The scaffolds are printed using common Material Extrusion (MEX) 3D printers and the limits, cost & reliability of the process are evaluated. The limits of standard MEX 3D-printing with off-the-shelf printer modifications is shown to achieve a minimum channel cross-section of 100×100 μm. The paper also lays out a protocol for the rapid fabrication of low-cost microfluidic channel moulds from the thermoplastic 3D-printed scaffolds, allowing the manufacture of customisable microfluidic systems without specialist equipment. The morphology of the resulting PDMS microchannels fabricated with the method are characterised and, when applied directly to glass, without plasma surface treatment, are shown to efficiently operate within the typical working pressures of commercial microfluidic devices. The technique is further validated through the demonstration of 2 common microfluidic devices; a fluid-mixer demonstrating the effective interconnecting scaffold design, and a microsphere droplet generator. The minimal cost of manufacture means that a 5000-piece physical library of mix-and-match channel scaffolds (100 μm scale) can be printed for ~$0.50 and made available to researchers and educators who lack access to appropriate technology. This simple yet innovative approach dramatically lowers the threshold for research and education into microfluidics and will make possible the rapid prototyping of point-of-care lab-on-a-chip diagnostic technology that is truly affordable the world over.

Boron nitride nanosheets functionalized channel scaffold favors microenvironment rebalance cocktail therapy for piezocatalytic neuronal repair

Abstract Persistent electrical stimulation by a mechanically actuated channel scaffold with porous and layered architecture is a promising strategy to facilitate neuronal microenvironment rebalance and locomotor recovery. Nevertheless, the relevant studies to date only reported preliminary findings but did not comprehensively evaluate its in-depth biological effects and mechanisms. To translate this smart scaffold into clinical application, biocompatibility and efficacy of the piezocatalytic nanoscaffold should be verified in vitro and in vivo. A novel ‘microenvironment rebalance cocktail therapy’ is proposed in this study. The piezoelectric boron nitride nanosheets (BNNS) functionalized polycaprolactone (PCL) channel scaffold displays high elasticity, hydrophilicity and biocompatibility, and stimulates cellular excretion of neurotrophic factors by increasing bioelectrical signal transduction under ultrasonic actuation in vitro. In addition, the scaffold modulates reactive oxygen species level and restores energy metabolic balance to maintain Schwann cell viability. The smart BNNS@PCL porous scaffold induces microvessel regrowth into neurons and reverses muscular atrophy after denervation in a severe sciatic nerve defect model in vivo. The design concept will be applicable in manufacture of an artificial bioelectrical system for advanced piezocatalytic nerve therapies.

Gelatin/Gelatin-modified nano hydroxyapatite composite scaffolds with hollow channel arrays prepared by extrusion molding for bone tissue engineering

Bone tissue engineering scaffold has been successfully applied in the field of bone repair, but the major limitation is the delivery of oxygen and nutrients throughout the bulk of engineered tissue, especially for large scaffolds. Researchers have found that scaffolds with hollow channels can effectively solve this problem. We utilized a novel extrusion approach to prepare scaffolds with hollow channel arrays, which has the advantages of economy, continuous production, and high efficiency. The hollow channel scaffolds were composed of gelatin and gelatin-modified nano hydroxyapatite (nHAP). X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), transmission electron microscopy (TEM) and atomic force microscope (AFM) results indicate that the prepared gelatin-modified nHAP are nanorod-like particles with the length of 30∼50 nm and width of 5∼15 nm. The nano particle is composed of crystalline HAP grains with the size less than 10 nm and amorphous phases. A part of OH− or are replaced by during preparation process, leading to the transformation of HAP into hydroxyl-carbonated apatite (HCA). The prepared hollow channel scaffolds exhibit much better mechanical properties compared with the sponge porous scaffolds. The axial compressive strength of the hollow channel scaffold with porosity of 51.3 ± 5.2% can reach 25 ± 1.4 MPa, which can meet the mechanical strength requirement as an implant completely. Moreover, the hollow channel scaffold exhibited good in vitro degradability and bioactivity. These results highlight the potential of using gelatin/gelatin-modified nHAP composite scaffolds with hollow channels for bone tissue engineering.

Direct Evidence for a Similar Molecular Mechanism Underlying Shaker Kv Channel Fast Inactivation and Clustering

The fast inactivation and clustering functions of voltage-dependent potassium channels play fundamental roles in electrical signaling. Recent evidence suggests that both these distinct channel functions rely on intrinsically disordered N- and C-terminal cytoplasmic segments that function as entropic clocks to time channel inactivation or scaffold protein-mediated clustering, both relying on what can be described as a “ball and chain” binding mechanism. Although the mechanisms employed in each case are seemingly analogous, both were put forward based on bulky chain deletions and further exhibit differences in reaction order. These considerations raised the question of whether the molecular mechanisms underlying Kv channel fast inactivation and clustering are indeed analogous. By taking a “chain”-level chimeric channel approach involving long and short spliced inactivation or clustering “chain” variants of the Shaker Kv channel, we demonstrate the ability of native inactivation and clustering “chains” to substitute for each other in a length-dependent manner, as predicted by the “ball and chain” mechanism. Our results thus provide direct evidence arguing that the two completely unrelated Shaker Kv channel processes of fast inactivation and clustering indeed occur according to a similar molecular mechanism.