Organ printing can be defined as layer-by-layer additive robotic biofabrication of three-dimensional functional living macrotissues and organ constructs using tissue spheroids as building blocks. The microtissues and tissue spheroids are living materials with certain measurable, evolving and potentially controllable composition, material and biological properties. Closely placed tissue spheroids undergo tissue fusion - a process that represents a fundamental biological and biophysical principle of developmental biology-inspired directed tissue self-assembly. It is possible to engineer small segments of an intraorgan branched vascular tree by using solid and lumenized vascular tissue spheroids. Organ printing could dramatically enhance and transform the field of tissue engineering by enabling large-scale industrial robotic biofabrication of living human organ constructs with "built-in" perfusable intraorgan branched vascular tree. Thus, organ printing is a new emerging enabling technology paradigm which represents a developmental biology-inspired alternative to classic biodegradable solid scaffold-based approaches in tissue engineering.

Organ Printing: Tissue Spheroids as Building Blocks

Over the past 20 years, and particularly in the last decade, significant developmental milestones have driven basic, translational, and clinical advances in the field of stem cell and regenerative medicine. In this article, we provide a systemic overview of the major recent discoveries in this exciting and rapidly developing field. We begin by discussing experimental advances in the generation and differentiation of pluripotent stem cells (PSCs), next moving to the maintenance of stem cells in different culture types, and finishing with a discussion of three-dimensional (3D) cell technology and future stem cell applications. Specifically, we highlight the following crucial domains: 1) sources of pluripotent cells; 2) next-generation in vivo direct reprogramming technology; 3) cell types derived from PSCs and the influence of genetic memory; 4) induction of pluripotency with genomic modifications; 5) construction of vectors with reprogramming factor combinations; 6) enhancing pluripotency with small molecules and genetic signaling pathways; 7) induction of cell reprogramming by RNA signaling; 8) induction and enhancement of pluripotency with chemicals; 9) maintenance of pluripotency and genomic stability in induced pluripotent stem cells (iPSCs); 10) feeder-free and xenon-free culture environments; 11) biomaterial applications in stem cell biology; 12) three-dimensional (3D) cell technology; 13) 3D bioprinting; 14) downstream stem cell applications; and 15) current ethical issues in stem cell and regenerative medicine. This review, encompassing the fundamental concepts of regenerative medicine, is intended to provide a comprehensive portrait of important progress in stem cell research and development. Innovative technologies and real-world applications are emphasized for readers interested in the exciting, promising, and challenging field of stem cells and those seeking guidance in planning future research direction.

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Advances in Pluripotent Stem Cells: History, Mechanisms, Technologies, and Applications.

Erratum : An Overview of Injectable Polymeric Hydrogels for Tissue Engineering

The diversity of manufacturing processes used to fabricate 3D implants, scaffolds, and tissue constructs is continuously increasing. This growing number of different applicable fabrication technologies include electrospinning, melt electrowriting, volumetric-, extrusion-, and laser-based bioprinting, the Kenzan method, and magnetic and acoustic levitational bioassembly, to name a few. Each of these fabrication technologies feature specific advantages and limitations, so that a combination of different approaches opens new and otherwise unreachable opportunities for the fabrication of hierarchical cell-material constructs. Ongoing challenges such as vascularization, limited volume, and repeatability of tissue constructs at the resolution required to mimic natural tissue is most likely greater than what one manufacturing technology can overcome. Therefore, the combination of at least two different manufacturing technologies is seen as a clear and necessary emerging trend, especially within biofabrication. This hybrid approach allows more complex mechanics and discrete biomimetic structures to address mechanotransduction and chemotactic/haptotactic cues. Pioneering milestone papers in hybrid fabrication for biomedical purposes are presented and recent trends toward future manufacturing platforms are analyzed.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/advs.201902953

Advances in Hybrid Fabrication toward Hierarchical Tissue Constructs.

Engineering spheroids potentiating cell-cell and cell-ECM interactions by self-assembly of stem cell microlayer.

Top-down tissue engineering aims to produce functional tissues using biomaterials as scaffolds, thus providing cues for cell proliferation and differentiation. Conversely, the bottom-up approach aims to precondition cells to form modular tissues units (building-blocks) represented by spheroids. In spheroid culture, adult stem cells are responsible for their extracellular matrix synthesis, re-creating structures at the tissue level. Spheroids from adult stem cells can be considered as organoids, since stem cells recapitulate differentiation pathways and also represent a promising approach for identifying new molecular targets (biomarkers) for diagnosis and therapy. Currently, spheroids can be used for scaffold-free (developmental engineering) or scaffold-based approaches. The scaffold promotes better spatial organization of individual spheroids and provides a defined geometry for their 3D assembly in larger and complex tissues. Furthermore, spheroids exhibit potent angiogenic and vasculogenic capacity and serve as efficient vascularization units in porous scaffolds for bone tissue engineering. An automated combinatorial approach that integrates spheroids into scaffolds is starting to be investigated for macro-scale tissue biofabrication.

https://www.mdpi.com/1422-0067/19/5/1285/pdf

Adult Stem Cells Spheroids to Optimize Cell Colonization in Scaffolds for Cartilage and Bone Tissue Engineering

Scaffold-free techniques in the developmental tissue engineering area are designed to mimic in vivo embryonic processes with the aim of biofabricating, in vitro, tissues with more authentic properties. Cell clusters called spheroids are the basis for scaffold-free tissue engineering. In this review, we explore the use of spheroids from adult mesenchymal stem/stromal cells as a model in the developmental engineering area in order to mimic the developmental stages of cartilage and bone tissues. Spheroids from adult mesenchymal stromal/stem cells lineages recapitulate crucial events in bone and cartilage formation during embryogenesis, and are capable of spontaneously fusing to other spheroids, making them ideal building blocks for bone and cartilage tissue engineering. Here, we discuss data from ours and other labs on the use of adipose stromal/stem cell spheroids in chondrogenesis and osteogenesis in vitro. Overall, recent studies support the notion that spheroids are ideal "building blocks" for tissue engineering by "bottom-up" approaches, which are based on tissue assembly by advanced techniques such as three-dimensional bioprinting. Further studies on the cellular and molecular mechanisms that orchestrate spheroid fusion are now crucial to support continued development of bottom-up tissue engineering approaches such as three-dimensional bioprinting.

Cartilage and bone tissue engineering using adipose stromal/stem cells spheroids as building blocks

Review question / Objective: Population: adults (aged between 18 and 50 years) with traumatic knee lesions who underwent treatment with mesenchymal stem cells; Intervention: defined by the treatment with mesenchymal stem cells; The comparison group: treatment with autologous chondrocytes or microfracture treatments; Primary outcome: formation of cartilage neo tissue in the defect area, determined by magnetic resonance imaging (MRI) or by direct visualization in second-look knee arthroscopy.; Secondary outcomes: based on clinical scores such as visual analog scale (VAS) for pain, Western Ontario and McMaster universities score (WOMAC), knee society score (KSS), Tegner and Lysholm.

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Renata Akemi Morais Matsui

Papers

Adult Stem Cells Spheroids to Optimize Cell Colonization in Scaffolds for Cartilage and Bone Tissue Engineering

Cartilage and bone tissue engineering using adipose stromal/stem cells spheroids as building blocks

Mesenchymal stem cells for the treatment of articular cartilage defects of the knee: an overview of systematic reviews