bims-cateng 2023-11-05 papers

bims-cateng

Biomed News

on Cell and tissue engineering

Issue of 2023–11–05
nine papers selected by
Chance Bowman, Dartmouth College

Efficient Large DNA Fragment Knock-in by Long dsDNA with 3'-Overhangs Mediated CRISPR Knock-in (LOCK) in Mammalian Cells.
Intelligent Vascularized 3D/4D/5D/6D-Printed Tissue Scaffolds.
Embedded 3D bioprinting - An emerging strategy to fabricate biomimetic & large vascularized tissue constructs.
Scaffold-based non-viral CRISPR delivery platform for efficient and prolonged gene activation to accelerate tissue regeneration.
Efficient Myogenic Activities Achieved through Blade-Casting-Assisted Bioprinting of Aligned Myoblasts Laden in Collagen Bioink.
Carbon Nanomaterial-Based Hydrogels as Scaffolds in Tissue Engineering: A Comprehensive Review.
A framework for ultra-low-input spatial tissue proteomics.
Applications of Scaffolds in Tissue Engineering: Current Utilization and Future Prospective.
Micropatterning the organization of multicellular structures in 3D biological hydrogels; insights into collective cellular mechanical interactions.

Bio Protoc. 2023 Oct 20. 13(20): e4853

Efficient Large DNA Fragment Knock-in by Long dsDNA with 3'-Overhangs Mediated CRISPR Knock-in (LOCK) in Mammalian Cells.

Wenjie Han, Haojun Liang, Jianqiang Bao.

  An efficient and precise genome-editing approach is in high demand in any molecular biology or cell biology laboratory worldwide. However, despite a recent rapid progress in the toolbox tailored for precise genome-editing, including the base editors and prime editors, there is still a need for a cost-effective knock-in (KI) approach amenable for long donor DNA cargos with high efficiency. By harnessing the high-efficient double-strand break (DSB) repair pathway of microhomology-mediated end joining, we previously showed that a specially designed 3'-overhang double-strand DNA (odsDNA) donor harboring 50-nt homology arm (HA) allows high-efficient exogenous DNA KI when combined with CRISPR-Cas9 technology. The lengths of the 3'-overhangs of odsDNA donors could be manipulated by the five consecutive phosphorothioate (PT) modifications. In this protocol, we detail the stepwise procedures to conduct the LOCK (Long dsDNA with 3'-Overhangs mediated CRISPR Knock-in) method for gene-sized (~1-3 kb) KI in mammalian cells.

Keywords:  CRISPR/Cas9; Cas9-PCV2 fusion; Gene knock-in (KI); Genome editing; Homologous recombination; dsDNA donor; odsDNA donor; ssDNA donor

DOI:  https://doi.org/10.21769/BioProtoc.4853
Nanomicro Lett. 2023 Oct 31. 15(1): 239

Intelligent Vascularized 3D/4D/5D/6D-Printed Tissue Scaffolds.

Xiaoyu Han, Qimanguli Saiding, Xiaolu Cai, Yi Xiao, Peng Wang, Zhengwei Cai, Xuan Gong, Weiming Gong, Xingcai Zhang, Wenguo Cui.

  Blood vessels are essential for nutrient and oxygen delivery and waste removal. Scaffold-repairing materials with functional vascular networks are widely used in bone tissue engineering. Additive manufacturing is a manufacturing technology that creates three-dimensional solids by stacking substances layer by layer, mainly including but not limited to 3D printing, but also 4D printing, 5D printing and 6D printing. It can be effectively combined with vascularization to meet the needs of vascularized tissue scaffolds by precisely tuning the mechanical structure and biological properties of smart vascular scaffolds. Herein, the development of neovascularization to vascularization to bone tissue engineering is systematically discussed in terms of the importance of vascularization to the tissue. Additionally, the research progress and future prospects of vascularized 3D printed scaffold materials are highlighted and presented in four categories: functional vascularized 3D printed scaffolds, cell-based vascularized 3D printed scaffolds, vascularized 3D printed scaffolds loaded with specific carriers and bionic vascularized 3D printed scaffolds. Finally, a brief review of vascularized additive manufacturing-tissue scaffolds in related tissues such as the vascular tissue engineering, cardiovascular system, skeletal muscle, soft tissue and a discussion of the challenges and development efforts leading to significant advances in intelligent vascularized tissue regeneration is presented.

Keywords:  Additive manufacturing; Intelligent; Osteogenesis; Tissue engineering; Vascularization

DOI:  https://doi.org/10.1007/s40820-023-01187-2
Bioact Mater. 2024 Feb;32 356-384

Embedded 3D bioprinting - An emerging strategy to fabricate biomimetic & large vascularized tissue constructs.

Harshavardhan Budharaju, Dhakshinamoorthy Sundaramurthi, Swaminathan Sethuraman.

  Three-dimensional bioprinting is an advanced tissue fabrication technique that allows printing complex structures with precise positioning of multiple cell types layer-by-layer. Compared to other bioprinting methods, extrusion bioprinting has several advantages to print large-sized tissue constructs and complex organ models due to large build volume. Extrusion bioprinting using sacrificial, support and embedded strategies have been successfully employed to facilitate printing of complex and hollow structures. Embedded bioprinting is a gel-in-gel approach developed to overcome the gravitational and overhanging limits of bioprinting to print large-sized constructs with a micron-scale resolution. In embedded bioprinting, deposition of bioinks into the microgel or granular support bath will be facilitated by the sol-gel transition of the support bath through needle movement inside the granular medium. This review outlines various embedded bioprinting strategies and the polymers used in the embedded systems with advantages, limitations, and efficacy in the fabrication of complex vascularized tissues or organ models with micron-scale resolution. Further, the essential requirements of support bath systems like viscoelasticity, stability, transparency and easy extraction to print human scale organs are discussed. Additionally, the organs or complex geometries like vascular constructs, heart, bone, octopus and jellyfish models printed using support bath assisted printing methods with their anatomical features are elaborated. Finally, the challenges in clinical translation and the future scope of these embedded bioprinting models to replace the native organs are envisaged.

Keywords:  Bioinks; Bioprinting; Complex bioprinting; Embedded bioprinting; Organ models bioprinting; Supportive bioprinting

DOI:  https://doi.org/10.1016/j.bioactmat.2023.10.012
Acta Biomater. 2023 Oct 30. pii: S1742-7061(23)00637-2. [Epub ahead of print]

Scaffold-based non-viral CRISPR delivery platform for efficient and prolonged gene activation to accelerate tissue regeneration.

Chuanxin Zhong, Shan He, Yuhong Huang, Jianfeng Yan, Junqin Wang, Wentao Liu, Ju Fang, Fuzeng Ren.

  Clustered regularly interspaced short palindromic repeat activation (CRISPRa) technology has emerged as a precise genome editing tool for activating endogenous transgene expression. While it holds promise for precise cell modification, its translation into tissue engineering has been hampered by biosafety concerns and suboptimal delivery methods. To address these challenges, we have developed a CRISPRa non-viral gene delivery platform by immobilizing non-viral CRISPRa complexes into a biocompatible hydrogel/nanofiber (Gel/NF) composite scaffold. The Gel/NF scaffold facilitates the controlled and sustained release of CRISPRa complexes and also promotes cell recruitment to the scaffold for efficient and localized transfection. As a proof of concept, we employed this CRISPRa delivery platform to activate the vascular endothelial growth factor (VEGF) gene in a rat model with full-thickness skin defects. Our results demonstrate sustained upregulation of VEGF expression even at 21 days post-implantation, resulting in enhanced angiogenesis and improved skin regeneration. These findings underscore the potential of the Gel/NF scaffold-based CRISPRa delivery platform as an efficient and durable strategy for gene activation, offering promising prospects for tissue regeneration. STATEMENT OF SIGNIFICANCE: Translation of clustered regularly interspaced short palindromic repeat activation (CRISPRa) therapy to tissue engineering is limited by biosafety concerns and unsatisfactory delivery strategy. To solve this issue, we have developed a CRISPRa non-viral gene delivery platform by immobilizing non-viral CRISPRa complexes into a biocompatible hydrogel/nanofiber (Gel/NF) composite scaffold. This scaffold enables controlled and sustained release of CRISPRa and can induce cell recruitment for localized transfection. As a proof of concept, we activated vascular endothelial growth factor (VEGF) in a rat model with full-thickness skin defects, leading to sustained upregulation of VEGF expression, enhanced angiogenesis and improved skin regeneration in vivo. These findings demonstrate the potential of this platform for gene activation, thereby offering promising prospects for tissue regeneration.

Keywords:  CRISPRa; gene delivery; gene therapy; tissue regeneration

DOI:  https://doi.org/10.1016/j.actbio.2023.10.029
Biomacromolecules. 2023 Nov 02.

Efficient Myogenic Activities Achieved through Blade-Casting-Assisted Bioprinting of Aligned Myoblasts Laden in Collagen Bioink.

SuHyeok Lee, WonJin Kim, GeunHyung Kim.

This study investigated mechanical stimulation combined with three-dimensional (3D) bioprinting as a new approach for introducing biophysical and biological cues for tissue regeneration. A blade-casting method in conjunction with bioprinting was employed to fabricate bioengineered skeletal muscle constructs using a bioink composed of C2C12 myoblasts and collagen type-I. Various printing process parameters were selected and optimized to achieve a highly organized cell alignment within the constructs. The resulting cell-aligned constructs demonstrated remarkable improvement in actin filament alignment and cell proliferation compared with conventionally printed cell-laden constructs. This improvement can be attributed to the synergistic effects of mechanotransduction, facilitating the cellular response to mechanical cues and the alignment of fibrillated collagen, which plays a significant role in modulating cellular functions and promoting muscle tissue regeneration. Furthermore, we assessed the impact of blade casting combined with 3D bioprinting on gene expression. The expression levels of myogenesis-related genes were substantially upregulated, with an approximately 1.6-fold increase compared to the constructs fabricated without the blade-casting technique. The results demonstrated the effectiveness of combining mechanical stimulation through blade casting with 3D bioprinting in promoting aligned cell structures, enhancing cellular functions, and driving muscle tissue regeneration.

DOI: https://doi.org/10.1021/acs.biomac.3c00749
Int J Nanomedicine. 2023 ;18 6153-6183

Carbon Nanomaterial-Based Hydrogels as Scaffolds in Tissue Engineering: A Comprehensive Review.

Thiago Domingues Stocco, Tianyi Zhang, Edgar Dimitrov, Anupama Ghosh, Alessandro Marcio Hakme da Silva, Wanessa C M A Melo, Willian Gonçalves Tsumura, André Diniz Rosa Silva, Gustavo F Sousa, Bartolomeu C Viana, Mauricio Terrones, Anderson Oliveira Lobo.

  Carbon-based nanomaterials (CBNs) are a category of nanomaterials with various systems based on combinations of sp2 and sp3 hybridized carbon bonds, morphologies, and functional groups. CBNs can exhibit distinguished properties such as high mechanical strength, chemical stability, high electrical conductivity, and biocompatibility. These desirable physicochemical properties have triggered their uses in many fields, including biomedical applications. In this review, we specifically focus on applying CBNs as scaffolds in tissue engineering, a therapeutic approach whereby CBNs can act for the regeneration or replacement of damaged tissue. Here, an overview of the structures and properties of different CBNs will first be provided. We will then discuss state-of-the-art advancements of CBNs and hydrogels as scaffolds for regenerating various types of human tissues. Finally, a perspective of future potentials and challenges in this field will be presented. Since this is a very rapidly growing field, we expect that this review will promote interdisciplinary efforts in developing effective tissue regeneration scaffolds for clinical applications.

Keywords:  biomaterial; carbon; nanotechnology; scaffold; tissue engineering

DOI:  https://doi.org/10.2147/IJN.S436867
Cell Syst. 2023 Oct 26. pii: S2405-4712(23)00287-9. [Epub ahead of print]

A framework for ultra-low-input spatial tissue proteomics.

Anuar Makhmut, Di Qin, Sonja Fritzsche, Jose Nimo, Janett König, Fabian Coscia.

  Spatial proteomics combining microscopy-based cell phenotyping with ultrasensitive mass-spectrometry-based proteomics is an emerging and powerful concept to study cell function and heterogeneity in (patho)physiology. However, optimized workflows that preserve morphological information for phenotype discovery and maximize proteome coverage of few or even single cells from laser microdissected tissue are currently lacking. Here, we report a robust and scalable workflow for the proteomic analysis of ultra-low-input archival material. Benchmarking in murine liver resulted in up to 2,000 quantified proteins from single hepatocyte contours and nearly 5,000 proteins from 50-cell regions. Applied to human tonsil, we profiled 146 microregions including T and B lymphocyte niches and quantified cell-type-specific markers, cytokines, and transcription factors. These data also highlighted proteome dynamics within activated germinal centers, illuminating sites undergoing B cell proliferation and somatic hypermutation. This approach has broad implications in biomedicine, including early disease profiling and drug target and biomarker discovery. A record of this paper's transparent peer review process is included in the supplemental information.

Keywords:  FFPE; deep visual proteomics; histopathology; mass spectrometry; proteomics; single-cell proteomics; spatial proteomics

DOI:  https://doi.org/10.1016/j.cels.2023.10.003
Curr Gene Ther. 2023 Oct 26.

Applications of Scaffolds in Tissue Engineering: Current Utilization and Future Prospective.

Shikha Yadav, Javed Khan, Agrima Yadav.

  Current regenerative medicine tactics focus on regenerating tissue structures pathologically modified by cell transplantation in combination with supporting scaffolds and biomolecules. Natural and synthetic polymers, bioresorbable inorganic and hybrid materials, and tissue decellularized were deemed biomaterials scaffolding because of their improved structural, mechanical, and biological abilities.Various biomaterials, existing treatment methodologies and emerging technologies in the field of Three-dimensional (3D) and hydrogel processing, and the unique fabric concerns for tissue engineering. A scaffold that acts as a transient matrix for cell proliferation and extracellular matrix deposition, with subsequent expansion, is needed to restore or regenerate the tissue. Diverse technologies are combined to produce porous tissue regenerative and tailored release of bioactive substances in applications of tissue engineering. Tissue engineering scaffolds are crucial ingredients. This paper discusses an overview of the various scaffold kinds and their material features and applications. Tabulation of the manufacturing technologies for fabric engineering and equipment, encompassing the latest fundamental and standard procedures.

Keywords:  Biomaterials; Hydrogels; Polymers; Scaffolds; Tissue engineering

DOI:  https://doi.org/10.2174/0115665232262167231012102837
Biofabrication. 2023 Oct 31.

Micropatterning the organization of multicellular structures in 3D biological hydrogels; insights into collective cellular mechanical interactions.

Bar Ergaz, Shahar Goren, Ayelet Lesman.

  Control over the organization of cells at the microscale level within supporting biomaterials can push forward the construction of complex tissue architectures for tissue engineering applications and enable fundamental studies of how tissue structure relates to its function. While cells patterning on 2D substrates is a relatively established and available procedure, micropatterning cells in biomimetic 3D hydrogels has been more challenging, especially with micro-scale resolution, and currently relies on sophisticated tools and protocols. We present a robust and accessible "peel-off" method to micropattern large arrays of individual cells or cell-clusters of precise sizes in biological 3D hydrogels, such as fibrin and collagen gels, with control over cell-cell separation distance and neighboring cells position. We further demonstrate partial control over cell position in the z-dimension by stacking two layers in varying distances between the layers. To demonstrate the potential of the micropatterning gel platform, we study the matrix-mediated mechanical interaction between array of cells that are accurately separated in defined distances. A collective process of intense cell-generated densified bands emerging in the gel between near neighbors was identified, along which cells preferentially migrate, a process relevant to tissue morphogenesis. The presented 3D gel micropatterning method can be used to reveal fundamental morphogenetic processes, and to reconstruct any tissue geometry with micrometer resolution in 3D biomimetic gel environments, leveraging the engineering of tissues in complex architectures.

Keywords:  Micropatterning; cell-matrix interaction; fibrous extracellular matrix; fibrous hydrogel; long-range cell-cell mechanical interaction; morphogenesis; traction force

DOI:  https://doi.org/10.1088/1758-5090/ad0849