Facile Fabrication of Three-Dimensional Hydrogel Film with Complex Tissue Morphology

In this study, we proposed a simple and easy method for fabricating a three-dimensional (3D) structure that can recapitulate the morphology of a tissue surface and deliver biological molecules into complex-shaped target tissues. To fabricate the 3D hydrogel film structure, we utilized a direct tissue casting method that can recapitulate tissue structure in micro-/macroscale using polydimethylsiloxane (PDMS). A replica 3D negative mold was manufactured by a polyurethane acrylate (PUA)-based master mold. Then, we poured the catechol-conjugated alginate (ALG-C) solution into the mold and evaporated it to form a dried film, followed by crosslinking the film using calcium chloride. The ALG-C hydrogel film had a tensile modulus of 725.2 ± 123.4 kPa and maintained over 95% of initial weight after 1 week without significant degradation. The ALG-C film captured over 4.5 times as much macromolecule (FITC-dextran) compared to alginate film (ALG). The cardiomyoblast cells exhibited high cell viability over 95% on ALG-C film. Moreover, the ALG-C film had about 70% of surface-bound lentivirus (1% in ALG film), which finally exhibited much higher viral transfection efficiency of GFP protein to C2C12 cells on the film than ALG film. In conclusion, we demonstrated a 3D film structure of biofunctionalized hydrogel for substrate-mediated drug delivery, and this approach could be utilized to recapitulate the complex-shaped tissues.

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3-D reconstruction of molecular shape from microcrystal thin sections

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100077311 ◽

1983 ◽

Vol 41 ◽

pp. 748-749

Author(s):

S. Cusack ◽

J.-C. Jésior

Keyword(s):

Three Dimensional ◽

Molecular Shape ◽

Biological Molecules ◽

Fourier Space ◽

Single Particles ◽

Thin Sections ◽

Standard Methods ◽

X Ray ◽

X Ray Crystallography ◽

Dimensional Reconstruction

Three-dimensional reconstruction techniques using electron microscopy have been principally developed for application to 2-D arrays (i.e. monolayers) of biological molecules and symmetrical single particles (e.g. helical viruses). However many biological molecules that crystallise form multilayered microcrystals which are unsuitable for study by either the standard methods of 3-D reconstruction or, because of their size, by X-ray crystallography. The grid sectioning technique enables a number of different projections of such microcrystals to be obtained in well defined directions (e.g. parallel to crystal axes) and poses the problem of how best these projections can be used to reconstruct the packing and shape of the molecules forming the microcrystal.Given sufficient projections there may be enough information to do a crystallographic reconstruction in Fourier space. We however have considered the situation where only a limited number of projections are available, as for example in the case of catalase platelets where three orthogonal and two diagonal projections have been obtained (Fig. 1).

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Methods for three-dimensional reconstruction of cellular components within a thick section

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100141871 ◽

1986 ◽

Vol 44 ◽

pp. 18-21

Author(s):

J. Frank ◽

B. F. McEwen ◽

M. Radermacher ◽

C. L. Rieder

Keyword(s):

Tilt Angle ◽

Tomographic Reconstruction ◽

3D Structure ◽

Three Dimensional ◽

Thick Section ◽

Three Dimensional Reconstruction ◽

Axis Ratio ◽

Dimensional Reconstruction ◽

Cellular Components

The tomographic reconstruction from multiple projections of cellular components, within a thick section, offers a way of visualizing and quantifying their three-dimensional (3D) structure. However, asymmetric objects require as many views from the widest tilt range as possible; otherwise the reconstruction may be uninterpretable. Even if not for geometric obstructions, the increasing pathway of electrons, as the tilt angle is increased, poses the ultimate upper limitation to the projection range. With the maximum tilt angle being fixed, the only way to improve the faithfulness of the reconstruction is by changing the mode of the tilting from single-axis to conical; a point within the object projected with a tilt angle of 60° and a full 360° azimuthal range is then reconstructed as a slightly elliptic (axis ratio 1.2 : 1) sphere.

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Cryomicroscopy of crotoxin complex crystals at 400 KV

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100156109 ◽

1989 ◽

Vol 47 ◽

pp. 826-827

Author(s):

Jaap Brink ◽

Wah Chiu

Keyword(s):

Signal To Noise Ratio ◽

3D Structure ◽

Three Dimensional ◽

Carbon Films ◽

Depth Of Field ◽

Basic Subunit ◽

Ewald Sphere ◽

Diffraction Patterns ◽

Complex Crystals ◽

Acidic Subunit

The crotoxin complex is a potent neurotoxin composed of a basic subunit (Mr = 12,000) and an acidic subunit (M = 10,000). The basic subunit possesses phospholipase activity whereas the acidic subunit shows no enzymatic activity at all. The complex's toxocity is expressed both pre- and post-synaptically. The crotoxin complex forms thin crystals suitable for electron crystallography. The crystals diffract up to 0.16 nm in the microscope, whereas images show reflections out to 0.39 nm2. Ultimate goal in this study is to obtain a three-dimensional (3D-) structure map of the protein around 0.3 nm resolution. Use of 100 keV electrons in this is limited; the unit cell's height c of 25.6 nm causes problems associated with multiple scattering, radiation damage, limited depth of field and a more pronounced Ewald sphere curvature. In general, they lead to projections of the unit cell, which at the desired resolution, cannot be interpreted following the weak-phase approximation. Circumventing this problem is possible through the use of 400 keV electrons. Although the overall contrast is lowered due to a smaller scattering cross-section, the signal-to-noise ratio of especially higher order reflections will improve due to a smaller contribution of inelastic scattering. We report here our preliminary results demonstrating the feasability of the data collection procedure at 400 kV.Crystals of crotoxin complex were prepared on carbon-covered holey-carbon films, quench frozen in liquid ethane, inserted into a Gatan 626 holder, transferred into a JEOL 4000EX electron microscope equipped with a pair of anticontaminators operating at −184°C and examined under low-dose conditions. Selected area electron diffraction patterns (EDP's) and images of the crystals were recorded at 400 kV and −167°C with dose levels of 5 and 9.5 electrons/Å, respectively.

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Detection, Averaging, and 3D Reconstruction of Biological Specimens on Hypercubes (Transputer-based) Computers

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100181026 ◽

1990 ◽

Vol 48 (1) ◽

pp. 454-455

Author(s):

Jose-Maria Carazo ◽

I. Benavides ◽

S. Marco ◽

J.L. Carrascosa ◽

E.L. Zapata

Keyword(s):

3D Reconstruction ◽

3D Structure ◽

Three Dimensional ◽

Software Tools ◽

Mapping Method ◽

Computer Architectures ◽

Parallel Computer ◽

Reconstruction Process ◽

Computing Power ◽

Order Of Magnitude

Obtaining the three-dimensional (3D) structure of negatively stained biological specimens at a resolution of, typically, 2 - 4 nm is becoming a relatively common practice in an increasing number of laboratories. A combination of new conceptual approaches, new software tools, and faster computers have made this situation possible. However, all these 3D reconstruction processes are quite computer intensive, and the middle term future is full of suggestions entailing an even greater need of computing power. Up to now all published 3D reconstructions in this field have been performed on conventional (sequential) computers, but it is a fact that new parallel computer architectures represent the potential of order-of-magnitude increases in computing power and should, therefore, be considered for their possible application in the most computing intensive tasks.We have studied both shared-memory-based computer architectures, like the BBN Butterfly, and local-memory-based architectures, mainly hypercubes implemented on transputers, where we have used the algorithmic mapping method proposed by Zapata el at. In this work we have developed the basic software tools needed to obtain a 3D reconstruction from non-crystalline specimens (“single particles”) using the so-called Random Conical Tilt Series Method. We start from a pair of images presenting the same field, first tilted (by ≃55°) and then untilted. It is then assumed that we can supply the system with the image of the particle we are looking for (ideally, a 2D average from a previous study) and with a matrix describing the geometrical relationships between the tilted and untilted fields (this step is now accomplished by interactively marking a few pairs of corresponding features in the two fields). From here on the 3D reconstruction process may be run automatically.

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A Novel Pseudomonas geniculata AGE Family Epimerase/Isomerase and Its Application in d-Mannose Synthesis

Foods ◽

10.3390/foods9121809 ◽

2020 ◽

Vol 9 (12) ◽

pp. 1809

Author(s):

Zhanzhi Liu ◽

Ying Li ◽

Jing Wu ◽

Sheng Chen

Keyword(s):

3D Structure ◽

Three Dimensional ◽

Optimal Temperature ◽

Reaction Conditions ◽

Enzymatic Production ◽

Physiological Properties ◽

Potential Applications ◽

Kinetics Of ◽

Optimal Reaction ◽

Gene Age

d-mannose has exhibited excellent physiological properties in the food, pharmaceutical, and feed industries. Therefore, emerging attention has been applied to enzymatic production of d-mannose due to its advantage over chemical synthesis. The gene age of N-acetyl-d-glucosamine 2-epimerase family epimerase/isomerase (AGEase) derived from Pseudomonas geniculata was amplified, and the recombinant P. geniculata AGEase was characterized. The optimal temperature and pH of P. geniculata AGEase were 60 °C and 7.5, respectively. The Km, kcat, and kcat/Km of P. geniculata AGEase for d-mannose were 49.2 ± 8.5 mM, 476.3 ± 4.0 s−1, and 9.7 ± 0.5 s−1·mM−1, respectively. The recombinant P. geniculata AGEase was classified into the YihS enzyme subfamily in the AGE enzyme family by analyzing its substrate specificity and active center of the three-dimensional (3D) structure. Further studies on the kinetics of different substrates showed that the P. geniculata AGEase belongs to the d-mannose isomerase of the YihS enzyme. The P. geniculata AGEase catalyzed the synthesis of d-mannose with d-fructose as a substrate, and the conversion rate was as high as 39.3% with the d-mannose yield of 78.6 g·L−1 under optimal reaction conditions of 200 g·L−1d-fructose and 2.5 U·mL−1P. geniculata AGEase. This novel P. geniculata AGEase has potential applications in the industrial production of d-mannose.

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A narrative overview of utilizing biomaterials to recapitulate the salient regenerative features of dental-derived mesenchymal stem cells

International Journal of Oral Science ◽

10.1038/s41368-021-00126-4 ◽

2021 ◽

Vol 13 (1) ◽

Author(s):

Sevda Pouraghaei Sevari ◽

Sahar Ansari ◽

Alireza Moshaverinia

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Tissue Regeneration ◽

3D Structure ◽

Three Dimensional ◽

Scientific Data ◽

Clinical Practices ◽

Human Teeth ◽

Innovative Strategies ◽

Local Recruitment

AbstractTissue engineering approaches have emerged recently to circumvent many limitations associated with current clinical practices. This elegant approach utilizes a natural/synthetic biomaterial with optimized physiomechanical properties to serve as a vehicle for delivery of exogenous stem cells and bioactive factors or induce local recruitment of endogenous cells for in situ tissue regeneration. Inspired by the natural microenvironment, biomaterials could act as a biomimetic three-dimensional (3D) structure to help the cells establish their natural interactions. Such a strategy should not only employ a biocompatible biomaterial to induce new tissue formation but also benefit from an easily accessible and abundant source of stem cells with potent tissue regenerative potential. The human teeth and oral cavity harbor various populations of mesenchymal stem cells (MSCs) with self-renewing and multilineage differentiation capabilities. In the current review article, we seek to highlight recent progress and future opportunities in dental MSC-mediated therapeutic strategies for tissue regeneration using two possible approaches, cell transplantation and cell homing. Altogether, this paper develops a general picture of current innovative strategies to employ dental-derived MSCs combined with biomaterials and bioactive factors for regenerating the lost or defective tissues and offers information regarding the available scientific data and possible applications.

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Midface Reconstruction: Planning and Outcome

Indian Journal of Plastic Surgery ◽

10.1055/s-0040-1721870 ◽

2020 ◽

Vol 53 (03) ◽

pp. 324-334

Author(s):

Gautam Biswas

Keyword(s):

Digital Imaging ◽

3D Structure ◽

Three Dimensional ◽

The Past ◽

Complex Anatomy ◽

Osseointegrated Implants ◽

Low Profile ◽

3D Printed ◽

Midface Reconstruction ◽

Reconstruction Planning

Abstract Reconstruction of the complex anatomy and aesthetics of the midface is often a challenge. A careful understanding of this three-dimensional (3D) structure is necessary. Anticipating the extent of excision and its planning following oncological resections is critical.In the past over two decades, with the advances in microsurgical procedures, contributions toward the reconstruction of this area have generated interest. Planning using digital imaging, 3D printed models, osseointegrated implants, and low-profile plates, has favorably impacted the outcome. However, there are still controversies in the management: to use single composite tissues versus multiple tissues; implants versus autografts; vascularized versus nonvascularized bone; prosthesis versus reconstruction.This article explores the present available options in maxillary reconstruction and outlines the approach in the management garnered from past publications and experiences.

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Identification of the conserved long non-coding RNAs in myogenesis

BMC Genomics ◽

10.1186/s12864-021-07615-0 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Anupam Bhattacharya ◽

Simang Champramary ◽

Tanya Tripathi ◽

Debajit Thakur ◽

Ilya Ioshikhes ◽

...

Keyword(s):

Gene Regulation ◽

Muscle Development ◽

Three Dimensional ◽

C2c12 Cells ◽

Mouse Muscle ◽

Rna Molecules ◽

Expression Of Genes ◽

Novel Approach ◽

Non Coding Rna ◽

Topologically Associated Domains

Abstract Background Our understanding of genome regulation is ever-evolving with the continuous discovery of new modes of gene regulation, and transcriptomic studies of mammalian genomes have revealed the presence of a considerable population of non-coding RNA molecules among the transcripts expressed. One such non-coding RNA molecule is long non-coding RNA (lncRNA). However, the function of lncRNAs in gene regulation is not well understood; moreover, finding conserved lncRNA across species is a challenging task. Therefore, we propose a novel approach to identify conserved lncRNAs and functionally annotate these molecules. Results In this study, we exploited existing myogenic transcriptome data and identified conserved lncRNAs in mice and humans. We identified the lncRNAs expressing differentially between the early and later stages of muscle development. Differential expression of these lncRNAs was confirmed experimentally in cultured mouse muscle C2C12 cells. We utilized the three-dimensional architecture of the genome and identified topologically associated domains for these lncRNAs. Additionally, we correlated the expression of genes in domains for functional annotation of these trans-lncRNAs in myogenesis. Using this approach, we identified conserved lncRNAs in myogenesis and functionally annotated them. Conclusions With this novel approach, we identified the conserved lncRNAs in myogenesis in humans and mice and functionally annotated them. The method identified a large number of lncRNAs are involved in myogenesis. Further studies are required to investigate the reason for the conservation of the lncRNAs in human and mouse while their sequences are dissimilar. Our approach can be used to identify novel lncRNAs conserved in different species and functionally annotated them.

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Biofabrication of Cell-Laden Gelatin Methacryloyl Hydrogels with Incorporation of Silanized Hydroxyapatite by Visible Light Projection

Polymers ◽

10.3390/polym13142354 ◽

2021 ◽

Vol 13 (14) ◽

pp. 2354

Author(s):

Jimmy Jiun-Ming Su ◽

Chih-Hsin Lin ◽

Hsuan Chen ◽

Shyh-Yuan Lee ◽

Yuan-Min Lin

Keyword(s):

Mechanical Properties ◽

Visible Light ◽

3D Structure ◽

Three Dimensional ◽

3D Cell Culture ◽

Composite Hydrogel ◽

Digital Light Processing ◽

Mg63 Cells ◽

Marrow Mesenchymal Stem Cells ◽

Low Cytotoxicity

Gelatin methacryloyl (GelMA) hydrogel is a photopolymerizable biomaterial widely used for three-dimensional (3D) cell culture due to its high biocompatibility. However, the drawback of GelMA hydrogel is its poor mechanical properties, which may compromise the feasibility of biofabrication techniques. In this study, a cell-laden GelMA composite hydrogel with a combination incorporating silanized hydroxyapatite (Si-HAp) and a simple and harmless visible light crosslinking system for this hydrogel were developed. The incorporation of Si-HAp into the GelMA hydrogel enhanced the mechanical properties of the composite hydrogel. Moreover, the composite hydrogel exhibited low cytotoxicity and promoted the osteogenic gene expression of embedded MG63 cells and Human bone marrow mesenchymal stem cells (hBMSCs). We also established a maskless lithographic method to fabricate a defined 3D structure under visible light by using a digital light processing projector, and the incorporation of Si-HAp increased the resolution of photolithographic hydrogels. The GelMA-Si-HAp composite hydrogel system can serve as an effective biomaterial in bone regeneration.

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