Template Scaffold Research Articles

Scaffold Pore Geometry Guides Gene Regulation and Bone-like Tissue Formation in Dynamic Cultures

Cells sense and respond to scaffold pore geometry and mechanical stimuli. Many fabrication methods used in bone tissue engineering render structures with poorly controlled pore geometries. Given that cell-scaffold interactions are complex, drawing a conclusion on how cells sense and respond to uncontrolled scaffold features under mechanical loading is difficult. In this study, monodisperse templated scaffolds (MTSC) were fabricated and used as well-defined porous scaffolds to study the effect of dynamic culture conditions on bone-like tissue formation. Human bone marrow-derived stromal cells were cultured on MTSC or conventional salt-leached scaffolds (SLSC) for up to 7 weeks, either under static or dynamic conditions (wall shear stress [WSS] using spinner flask bioreactors). The influence of controlled spherical pore geometry of MTSC subjected to static or dynamic conditions on osteoblast cell differentiation, bone-like tissue formation, structure, and distribution was investigated. WSS generated within the two idealized geometrical scaffold features was assessed. Distinct response to fluid flow in osteoblast cell differentiation were shown to be dependent on scaffold pore geometry. As revealed by collagen staining and microcomputed tomography images, dynamic conditions promoted a more regular extracellular matrix (ECM) formation and mineral distribution in both scaffold types compared with static conditions. The results showed that regulation of bone-related genes and the amount and the structure of mineralized ECM were dependent on scaffold pore geometry and the mechanical cues provided by the two different culture conditions. Under dynamic conditions, SLSC favored osteoblast cell differentiation and ECM formation, whereas MTSC enhanced ECM mineralization. The spherical pore shape in MTSC supported a more trabecular bone-like structure under dynamic conditions compared with MTSC statically cultured or to SLSC under either static or dynamic conditions. These results suggest that cell activity and bone-like tissue formation is driven not only by the pore geometry but also by the mechanical environment. This should be taken into account in the future design of complex scaffolds, which should favor cell differentiation while guiding the formation, structure, and distribution of the engineered bone tissue. This could help to mimic the anatomical complexity of the bone tissue structure and to adapt to each bone defect needs. Impact statement Aging of the human population leads to an increasing need for medical implants with high success rate. We provide evidence that cell activity and the amount and structure of bone-like tissue formation is dependent on the scaffold pore geometry and on the mechanical environment. Fabrication of complex scaffolds comprising concave and planar pore geometries might represent a promising direction toward the tunability and mimicry the structural complexity of the bone tissue. Moreover, the use of fabrication methods that allow a systematic fabrication of reproducible and geometrically controlled structures would simplify scaffold design optimization.

Gradient scaffold with spatial growth factor profile for osteochondral interface engineering

Osteochondral (OC) matrix design poses a significant engineering challenge due to the complexity involved with bone-cartilage interfaces. To better facilitate the regeneration of OC tissue, we developed and evaluated a biodegradable matrix with uniquely arranged bone and cartilage supporting phases: a poly(lactic-co-glycolic) acid (PLGA) template structure with a porosity gradient along its longitudinal axis uniquely integrated with hyaluronic acid hydrogel. Micro-CT scanning and imaging confirmed the formation of an inverse gradient matrix. Hydroxyapatite was added to the PLGA template which was then plasma-treated to increase hydrophilicity and growth factor affinity. An osteogenic growth factor (bone morphogenetic protein 2; BMP-2) was loaded onto the template scaffold via adsorption, while a chondrogenic growth factor (transforming growth factor beta 1; TGF-β1) was incorporated into the hydrogel phase. Confocal microscopy of the growth factor loaded matrix confirmed the spatial distribution of the two growth factors, with chondrogenic factor confined to the cartilaginous portion and osteogenic factor present throughout the scaffold. We observed spatial differentiation of human mesenchymal stem cells (hMSCs) into cartilage and bone cells in the scaffolds in vitro: cartilaginous regions were marked by increased glycosaminoglycan production, and osteogenesis was seen throughout the graft by alizarin red staining. In a dose-dependent study of BMP-2, hMSC pellet cultures with TGF-β1 and BMP-2 showed synergistic effects on chondrogenesis. These results indicate that development of an inverse gradient matrix can spatially distribute two different growth factors to facilitate chondrogenesis and osteogenesis along different portions of a scaffold, which are key steps needed for formation of an OC interface.

Development and Angiogenic Potential of Cell-Derived Microtissues Using Microcarrier-Template.

Tissue engineering and regenerative medicine approaches use biomaterials in combination with cells to regenerate lost functions of tissues and organs to prevent organ transplantation. However, most of the current strategies fail in mimicking the tissue’s extracellular matrix properties. In order to mimic native tissue conditions, we developed cell-derived matrix (CDM) microtissues (MT). Our methodology uses poly-lactic acid (PLA) and Cultispher® S microcarriers’ (MCs’) as scaffold templates, which are seeded with rat bone marrow mesenchymal stem cells (rBM-MSCs). The scaffold template allows cells to generate an extracellular matrix, which is then extracted for downstream use. The newly formed CDM provides cells with a complex physical (MT architecture) and biochemical (deposited ECM proteins) environment, also showing spontaneous angiogenic potential. Our results suggest that MTs generated from the combination of these two MCs (mixed MTs) are excellent candidates for tissue vascularization. Overall, this study provides a methodology for in-house fabrication of microtissues with angiogenic potential for downstream use in various tissue regenerative strategies.

Modern concepts of the osteogenic potential of mesenchymal stem cells and the creation of bioengineering structures for bone tissue repair

The following review summarizes the latest studies on in vitro osteogenic mesenchymal stem cell differentiation and selection of scaffolds that can maintain the viability and functional activity of these cells for bone tissue repair. In the last time, there have been investigated a lot of issues such as the stimulation and development osteogenic differentiation of MSCs, the growth factors – inducers of osteogenesis in MSCs, the creation of 3D constructions of cells in different scaffolds. A deeper understanding of the osteogenic differentiation mechanisms can result in the novel therapeutic opportunities of bone disease treatment. Special attention is given to materials for scaffold designs and template–cell interactions, which is of great importance for the structuring and functioning of an engineered tissue.

Uniform 40-µm-pore diameter precision templated scaffolds promote a pro-healing host response by extracellular vesicle immune communication.

Implanted porous precision templated scaffolds (PTS) with 40-µm spherical pores reduce inflammation and foreign body reaction (FBR) while increasing vascular density upon implantation. Larger or smaller pores, however, promote chronic inflammation and FBR. While macrophage (MØ) recruitment and polarization participates in perpetuating this pore-size-mediated phenomenon, the driving mechanism of this unique pro-healing response is poorly characterized. We hypothesized that the primarily myeloid PTS resident cells release small extracellular vesicles (sEVs) that induce pore-size-dependent pro-healing effects in surrounding T cells. Upon profiling resident immune cells and their sEVs from explanted 40-µm- (pro-healing) and 100-µm-pore diameter (inflammatory) PTS, we found that PTS pore size did not affect PTS resident immune cell population ratios or the proportion of myeloid sEVs generated from explanted PTS. However, quantitative transcriptomic assessment indicated cell and sEV phenotype were pore size dependent. In vitro experiments demonstrated the ability of PTS cell-derived sEVs to stimulate T cells transcriptionally and proliferatively. Specifically, sEVs isolated from cells inhabiting explanted 100 μm PTS significantly upregulated Th1 inflammatory gene expression in immortalized T cells. sEVs isolated from cell inhabiting both 40- and 100-μm PTS upregulated essential Treg transcriptional markers in both primary and immortalized T cells. Finally, we investigated the effects of Treg depletion on explanted PTS resident cells. FoxP3+ cell depletion suggests Tregs play a unique role in balancing T cell subset ratios, thus driving host response in 40-μm PTS. These results indicate that predominantly 40-µm PTS myeloid cell-derived sEVs affect T cells through a distinct, pore-size-mediated modality.

In Vitro Reconstitution of Human Mitochondrial Transcription.

In vitro assay based on a reconstituted mitochondrial transcription system serves as a method of choice to probe the functional importance of proteins and their structural motifs. Here we describe protocols for transcription assays designed to probe activity of the human mitochondrial RNA polymerase and the transcription initiation complex using RNA-DNA scaffold and synthetic promoter templates.

In Situ Liquid Cell Transmission Electron Microscopy Observation of Dynamic Process of Oleic Acid Emulsion with Gold Nanorods

Oleic acid (OA) emulsion is widely used in nanomaterial synthesis and applications. The emulsion droplets can serve as template scaffolds for nanoparticle self-organization, while adding nanopartic...

Evolutionary selection of a 19-stranded mitochondrial β-barrel scaffold bears structural and functional significance

Transmembrane β-barrels of eukaryotic outer mitochondrial membranes (OMMs) are major channels of communication between the cytosol and mitochondria and are indispensable for cellular homeostasis. A structurally intriguing exception to all known transmembrane β-barrels is the unique odd-stranded, i.e. 19-stranded, structures found solely in the OMM. The molecular origins of this 19-stranded structure and its associated functional significance are unclear. In humans, the most abundant OMM transporter is the voltage-dependent anion channel. Here, using the human voltage-dependent anion channel as our template scaffold, we designed and engineered odd- and even-stranded structures of smaller (V216, V217, V218) and larger (V220, V221) barrel diameters. Determination of the structure, dynamics, and energetics of these engineered structures in bilayer membranes reveals that the 19-stranded barrel surprisingly holds modest to low stability in a lipid-dependent manner. However, we demonstrate that this structurally metastable protein possesses superior voltage-gated channel regulation, efficient mitochondrial targeting, and in vivo cell survival, with lipid-modulated stability, all of which supersede the occurrence of a metastable 19-stranded scaffold. We propose that the unique structural adaptation of these transmembrane transporters exclusively in mitochondria bears strong evolutionary basis and is functionally significant for homeostasis.

From orexin receptor agonist YNT-185 to novel antagonists with drug-like properties for the treatment of insomnia

YNT-185 is the first known small molecule acting as orexin 2 receptor (OX2R) agonist with implication to narcolepsy treatment, served as a template scaffold in generating a small set of seven compounds with predictive affinity to OX2R. The design of the new small molecules was driven mostly by improving physicochemical properties of the parent drug YNT-185 in parallel with in silico studies, later suggesting their favorable binding modes within the active site of OX2R. We obtained seven new potential OX2R binders that were evaluated in vitro for their CNS availability, cytotoxicity, and behavior pattern on OX2R. Out of them, 15 emerged as the most potent modulator of OX2R, which, contrary to YNT-185, displayed inverse mode of action, i.e. antagonist profile. 15 was also submitted to an in vivo experiment revealing its ability to permeate through BBB into the brain with a short half-life.

Basic Principles of Emulsion Templating and Its Use as an Emerging Manufacturing Method of Tissue Engineering Scaffolds.

Tissue engineering (TE) aims to regenerate critical size defects, which cannot heal naturally, by using highly porous matrices called TE scaffolds made of biocompatible and biodegradable materials. There are various manufacturing techniques commonly used to fabricate TE scaffolds. However, in most cases, they do not provide materials with a highly interconnected pore design. Thus, emulsion templating is a promising and convenient route for the fabrication of matrices with up to 99% porosity and high interconnectivity. These matrices have been used for various application areas for decades. Although this polymer structuring technique is older than TE itself, the use of polymerised internal phase emulsions (PolyHIPEs) in TE is relatively new compared to other scaffold manufacturing techniques. It is likely because it requires a multidisciplinary background including materials science, chemistry and TE although producing emulsion templated scaffolds is practically simple. To date, a number of excellent reviews on emulsion templating have been published by the pioneers in this field in order to explain the chemistry behind this technique and potential areas of use of the emulsion templated structures. This particular review focusses on the key points of how emulsion templated scaffolds can be fabricated for different TE applications. Accordingly, we first explain the basics of emulsion templating and characteristics of PolyHIPE scaffolds. Then, we discuss the role of each ingredient in the emulsion and the impact of the compositional changes and process conditions on the characteristics of PolyHIPEs. Afterward, current fabrication methods of biocompatible PolyHIPE scaffolds and polymerisation routes are detailed, and the functionalisation strategies that can be used to improve the biological activity of PolyHIPE scaffolds are discussed. Finally, the applications of PolyHIPEs on soft and hard TE as well as in vitro models and drug delivery in the literature are summarised.

Self-standing zeolite foam monoliths with hierarchical micro-meso-macroporous structures.

The zeolite monoliths were synthesized by a facile polymer scaffold template assisted hydrothermal method. The selected foam-shaped template of a polyurethane (PU) foam monolith, was used to prepare the self-standing zeolite foam (ZF) monolithic materials. The obtained ZF products can preserve the same size, shape and macroporous network structure of the original PU foam scaffold template, although the zeolite nano-crystallites had been fully substituted for the PU template to form the new skeleton struts and walls. The as-synthesized ZF products demonstrated abundant hierarchical porosity (involving triple micro-, meso- and macropores). Meanwhile, compared with the conventional zeolite powders, the self-standing ZF monolithic materials exhibited greater total pore volume and nearly three times higher mesopore volume, suggesting wider applications as catalysts, catalyst supports and adsorbents in industry.

Graphene Foam (GF)/Manganese Oxide (MnO2) Nanocomposites for High Performance Supercapacitors

Abstract We report supercapacitive characteristics of hierarchically porous architectured graphene foam (GF)/manganese oxide (MnO2) nanocomposites fabricated via a simple two-step synthesis route. A polyurethane (PU) foam with graphene flakes adsorbed onto the pore walls is pyrolyzed to obtain GF. The MnO2 is then electrochemically deposited onto GF to form a GF/MnO2 (with a conductivity of 2.85 and 1.1 Sm−1, respectively) nanocomposite. The GF is an ideal scaffold template for electrochemical deposition of MnO2 and fast ion transport from MnO2 to GF. The GF/MnO2 supercapacitor shows excellent specific capacitance (672 F g−1), rate capability (85% capacity retention over 1 to 20 A g−1), and long cycle life (2% capacity loss after 10000 charge/discharge cycles) for 0.5 mg cm−2 MnO2 loading. Furthermore, the optimized supercapacitor delivers a specific energy of 95 Wh kg−1 at a specific power of 1 kW kg−1, which is substantially higher than that of most of the reported GF based supercapacitors. The results obtained here may pave the way for the development of commercial supercapacitors for future energy storage systems.

In-situ deposition of three-dimensional graphene on selective laser melted copper scaffolds for high performance applications

Currently, three-dimensional graphene (3DG) fabrication was restricted by the complicated process, strict chemical reactions as well as structural accuracy. Herein we creatively propose a bottom-up strategy that leverages the selective laser melting (SLM) technique to manufacture a three-dimensional (3D) porous copper template. Graphene was then in-situ grown via chemical vapor deposition (CVD) on the obtained porous Cu template, forming 3DG composites. A combination of conventional graphene growth via CVD technique with SLM fabricated scaffold templates enabled an accurate design and regulation of 3DG from macro-structure (unit type, porosity, aperture) to micro-structure (texture, surface quality) through an elaborately manipulated porous copper scaffold. The 3DG/copper scaffold could achieve around 88% and 27% enhancement in electromagnetic interference (EMI) shielding and thermal diffusion, respectively. Particularly, the highest EMI shielding efficiency (SE) can reach up to 47.8 dB at 2.7 GHz and exhibit an average SE of 32.3 dB at the range of 2–18 GHz. The synergistic shielding mechanisms accounted for the improvement derived from the use of hybrid composite materials and precise architecture of the SLM porous structure.

Nickel-copper graphene foam prepared by atmospheric pressure chemical vapour deposition for supercapacitor applications

This study reports on Ni and NiCu graphene foam prepared by atmospheric pressure chemical vapour deposition. The NiCu foam alloys were prepared using a three-dimensional (3D) scaffold template of polycrystalline nickel foam which is commonly used as a current collector in supercapacitor electrodes. The morphological, composition and structural characterization of as-grown Ni and NiCu graphene foam were carried out using confocal Raman spectroscopy imaging, scanning electron microscopy (SEM), high-resolution transmission electron microscopy (HR-TEM), energy-dispersive X-ray spectrometer (EDS) and X-ray photoelectron spectroscopy (XPS). The results demonstrated that NiCu graphene foam is mainly monolayer and bilayer as compared to Ni graphene foam which is typically a few-layer graphene. The electrochemical performance of Ni and NiCu graphene foam as current collectors were evaluated in a three-electrode electrochemical cell using 1 M NaSO4 aqueous electrolyte and porous activated carbon (AC) produced from lightweight material cork (Quercus suber) as the active electrode material. The AC on NiCu graphene foam current collectors exhibited a high specific capacitance (250–300 F g−1 at 1 A g−1) and good electrochemical stability (94–100% capacitance retention) over 6000 constant charge-discharge cycles at 5 A g−1.

A standalone bioreactor system to deliver compressive load under perfusion flow to hBMSC-seeded 3D chitosan-graphene templates

The availability of engineered biological tissues holds great potential for both clinical applications and basic research in a life science laboratory. A prototype standalone perfusion/compression bioreactor system was proposed to address the osteogenic commitment of stem cells seeded onboard of 3D chitosan-graphene (CHT/G) templates. Testing involved the coordinated administration of a 1 mL/min medium flow rate together with dynamic compression (1% strain at 1 Hz; applied twice daily for 30 min) for one week. When compared to traditional static culture conditions, the application of perfusion and compression stimuli to human bone marrow stem cells using the 3D CHT/G template scaffold induced a sizable effect. After using the dynamic culture protocol, there was evidence of a larger number of viable cells within the inner core of the scaffold and of enhanced extracellular matrix mineralization. These observations show that our novel device would be suitable for addressing and investigating the osteogenic phenotype commitment of stem cells, for both potential clinical applications and basic research.

Human Dental Pulp Stem Cells: recent findings and current research

Abstract Prevalence of neurodegenerative diseases, most of which are life threatening and incurable, is an increasing clinical problem. To date, studies have demonstrated a superior proliferation rate of dental pulp stem cells (DPSCs) compared to other mesenchymal stem cells in vitro. DPSCs has recently been recognized as a novel treatment strategy for neurodegenerative disease, due to their advanced potential for neurogenic differentiation. The oral cavity has been described as a promising source of dental pulp stem cells. DPSCs are widely used in regenerative dentistry holding alternative capacity for osteogenic differentiation and therefore new promises for tissue and whole tooth regeneration. Dental stem cell banking offers a plentiful source of stem cells representing great potential for cell reprogramming and thus cell therapy. Recently, the association of pulp stem cells with three – dimensional scaffold templates allows for building up naturally derived implants. This review introduces to unique properties of DPSCs and biological factors influencing mineralization, proliferation and differentiation of pulp stem cells. Latest research studies are compared in terms of effectiveness and limitations of techniques for the isolation of pulp stem cells, including the enzymatic digestion and the explant culture methods. Moreover, a short overview of most recent findings and clinical application of DPSCs is proffered including progress of current research and limitations still to be addressed in the nearest future. Finally, the article presents new advances in the area of regenerative dentistry and regenerative medicine, including three dimensional printing and three dimensional analysis, emerged to deepen studies under procedures to replace the non patient specific artificial implants. Running title: DPSCs - review

Polypyrrole-Coated Polycaprolactone-Gelatin Conductive Nanofibers: Fabrication and Characterization

Electrically conductive nanofibrous meshes composed of polypyrrole, polycaprolactone and gelatin construct using a two-step approach including electrospinning and in situ polymerization. Nanometer-scale thick polypyrrole (PPy) is grown on the surface of electrospun polycaprolactone-gelatin (PCL-Gel) nanofiber by in situ chemical polymerization. Different PCL-to-gel ratios and fiber orientations are employed to construct template scaffolds for the coating reaction. SEM analysis indicates the fiber diameter of 162–207 nm and the coating thickness of 30–93 nm. Porosity estimation confirms conservation of total porosity in the coated mats. ATR-FTIR of the composite reveals that functional groups of all three polymers partially exist on the surface. Furthermore, XRD analysis verifies the crystalline nature of the hybrid scaffold. High electrical conductivity (4.6–5.8 S/cm) and convenient mechanical properties of the coated membranes are further achieved. The collective information of the current investigation introduces suitable potential candidates for diverse biomedical applications.

The effect of alloying elements on the properties of pressed and non-pressed biodegradable Fe–Mn–Ag powder metallurgy alloys

Current trends in the biodegradable scaffold industry call for powder metallurgy methods in which compression cannot be applied due to the nature of the scaffold template itself and the need to retain the shape of an underlying template throughout the fabrication process. Iron alloys have been shown to be good candidates for biomedical applications where load support is required. Fe–Mn alloys were researched extensively for this purpose. Current research shows that all metallurgical characterisation and corrosion test on Fe–Mn and Fe–Mn–Ag non pre-alloyed powder alloys are performed on alloys which are initially pressed into greens and subsequently sintered. In order to combine the cutting-edge field of biodegradable metallic alloys with scaffold production, metallurgical characterisation of pressed and non-pressed Fe, Fe–Mn and Fe–Mn–Ag sintered elemental powder compacts was carried out in this study. This was performed along with determination of the corrosion rate of the same alloys in in vitro mimicking solutions. These solutions were synthesised to mimic the osteo environment in which the final scaffolds are to be used.Both pressed and non-pressed alloys formed an austenite phase under the right sintering conditions. The corrosion rate of the non-pressed alloy was greater than that of its pressed counterpart. In a potentiodynamic testing scenario, addition of silver to the alloy formed a separate silver phase which galvanically increased the corrosion rate of the pressed alloy. This result wasn't replicated in the non-pressed alloys in which the corrosion rate was seen to remain similar to the non-silver-bearing alloy counterparts.

MOF-Derived Co,N Codoped Carbon/Ti Mesh Counter Electrode for High-Efficiency Quantum Dot Sensitized Solar Cells.

Carbon supported on titanium mesh electrodes has been recognized as the best performing counter electrodes (CEs) in quantum dot sensitized solar cells (QDSCs). Herein, layered double hydroxides (LDHs) are applied as a scaffold template for the growth of cobalt-zeolite-imidazole framework (ZIF-67) crystals, and micrometer-sized Co,N codoped porous carbon materials (Co,N-C) are obtained through a carbonization process. The as-prepared Co,N-C exhibits favorable features for electrocatalytic reduction of polysulfide, including a high surface area of 491.36 m2/g, highly effective active sites, and a hierarchical micro/mesoporous structure. Due to the large particle size, the obtained Co,N-C can couple with a Ti mesh substrate for the fabrication of high-performance Co,N-C/Ti CEs for QDSCs. As a result, the corresponding QDSCs exhibit an average efficiency of 13.55% (Jsc = 25.93 mA/cm2, Voc = 0.778 V, FF = 0.672), which is a 10.5% enhancement compared to the previous best result from the N-doped mesoporous carbon counterpart.

Discovery of SBF1 as an allosteric inhibitor targeting the PIF-pocket of 3-phosphoinositide-dependent protein kinase-1.

3-phosphoinositide-dependent protein kinase-1 (PDK1) plays a crucial role in the signal transduction of massive growth-related protein kinases. In this work, a computational study has been performed to investigate the binding pose of the hydrolyzed product of SBF1 (SBF1-) with PDK1. The binding pose was predicted by Vina and was further refined in a molecular dynamics simulation. For comparison, four published low molecular weight compounds (PS48, PS171, PS182, and PS210) binding with PDK1 were also studied. SBF1- was anchored in the PIF-pocket of PDK1 with salt bridge interaction using its carboxylate moiety, which is a common feature among the known ligands. Hydrogen bonds to THR148 and vdW interactions with GLN150 also have contributions to the association affinity. The allosteric regulation on PDK1 via the binding of SBF1- was further addressed. The binding affinity of SBF1- in complex with PDK1 is comparable to those of PS171 and PS182, with an estimated IC50 in a range from 2.0 to 10.0 μ molar. Comparison between the free energy profiles with the presence or absence of SBF1- in the binding pocket indicates that the binding of SBF1- enhances the hinge motion and suppresses the fluctuation of the end-to-end distance in α B of PDK1. These results demonstrate that SBF1- is a promising allosteric regulator of PDK1 targeting the PIF-binding pocket and can serve as a new scaffold template for the design of new drugs targeting PDK1.