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Formation and morphology change of pTSs. (a) Dimensions of alginate capsules (n = 3). (b) Homogenous distribution of cells and porogens in the mixture, with red arrows indicating the porogens and an image with higher magnification showing well-defined porogens surrounded by cells. (c) Morphology change of microinjected cells in the alginate capsule before (c1)–(c3) and after (c4)–(c7) de-crosslinking. (d) Diameter change of pTSs overtime (n = 3).
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The scalability of cell aggregates such as spheroids, strands, and rings has been restricted by diffusion of nutrient and oxygen into their core. In this study, we introduce a novel concept in generating tissue building blocks with micropores, which represents an alternative solution for vascularization. Sodium alginate porogens were mixed with hum...
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Bone/cartilage repair and regeneration have been popular and difficult issues in medical research. Tissue engineering is rapidly evolving to provide new solutions to this problem, and the key point is to design the appropriate scaffold biomaterial. In recent years，microsphere-based scaffolds have been considered suitable scaffold materials for bone...
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... In the presence of cyclic stretching, the expression of tendon-specific proteins and cellular orientation in the tissue strands exhibited significant enhancement. Moreover, to improve the cell viability of scaled-up cell aggregates with diameters over 500 μm, sodium alginate porogens were blended with human ADSCs to generate tissue building blocks with micropores, which were termed porous tissue strands (pTSs) [113]. Compared with their solid counterparts, pTSs enabled the perfusion of oxygen and nutrients to the deeper region, enhanced cellular viability, and promoted tissue maturation for the long term, which represented an alternative solution for vascularization (Fig. 14d). ...
... Part (c) was adapted from [112], Copyright 2023, with permission from the American Chemical Society. Part (d) was adapted from [113], Copyright 2018, with permission from IOP Publishing single-material or single-cell bioinks are not well suited to the replication of these intricate microenvironments. Therefore, the development of multimaterial and multicellular VBP is essential for accurately simulating the complex microenvironment of artificial organs. ...
As surgical procedures transition from conventional resection to advanced tissue-regeneration technologies, human disease therapy has witnessed a great leap forward. In particular, three-dimensional (3D) bioprinting stands as a landmark in this setting, by promising the precise integration of biomaterials, cells, and bioactive molecules, thus opening up a novel avenue for tissue/organ regeneration. Curated by the editorial board of Bio-Design and Manufacturing, this review brings together a cohort of leading young scientists in China to dissect the core functionalities and evolutionary trajectory of 3D bioprinting, by elucidating the intricate challenges encountered in the manufacturing of transplantable organs. We further delve into the translational pathway from scientific research to clinical application, emphasizing the imperativeness of establishing a regulatory framework and rigorously enforcing quality-control measures. Finally, this review outlines the strategic landscape and innovative achievements of China in this field and provides a comprehensive roadmap for researchers worldwide to propel this field collectively to even greater heights.
... Alginate was also prepared in the form of microspheres to deliver growth factors, proteins, and drugs in tissue engineering [114] . Inspired by the advantage of the alginate microsphere, Wu et al. embedded the alginate microspheres within the cell aggregates to generate porous tissue strands with high cell density [116] . The incorporation of alginate microspheres facilitated the permeation of oxygen and nutrition, promoting cell viability within the cell aggregates, which offers new insight into scaffold-free biofabrication. ...
The major apparatuses used for three-dimensional (3D) bioprinting include extrusion-based, droplet-based, and laser-based bioprinting. Numerous studies have been proposed to fabricate bioactive 3D bone tissues using different bioprinting techniques. In addition to the development of bioinks and assessment of their printability for corresponding bioprinting processes, in vitro and in vivo success of the bioprinted constructs, such as their mechanical properties, cell viability, differentiation capability, immune responses, and osseointegration, have been explored. In this review, several major considerations, challenges, and potential strategies for bone bioprinting have been deliberated, including bioprinting apparatus, biomaterials, structure design of vascularized bone constructs, cell source, differentiation factors, mechanical properties and reinforcement, hypoxic environment, and dynamic culture. In addition, up-to-date progress in bone bioprinting is summarized in detail, which uncovers the immense potential of bioprinting in re-establishing the 3D dynamic microenvironment of the native bone. This review aims to assist the researchers to gain insights into the reconstruction of clinically relevant bone tissues with appropriate mechanical properties and precisely regulated biological behaviors.
... Scaffold-free bioprinting approach [15], on the other hand, focuses on tissue fabrication using cell aggregates, without the need for scaffold support, triggering cells to secrete their own extracellular matrix (ECM). Cell aggregates can be formed into geometrical configurations, for example, strands, honeycomb, or spheroids, and then allowed to fuse into larger tissues [16][17][18]. Both approaches have pros and cons, and in cases of 3D bioprinting, they may complement each other in the pursuit of meeting the ever-increasing demand for fabrication of scalable physically-relevant tissues or organs. ...
... Larger spheroids possess more dead cells in their core due to hypoxia and insufficient nutrients infusion. In addition, percent live cells often cannot be quantified with a non-destructive measurement assay easily [16]. This heterogeneous makeup of dead and live cells in the core and shell, respectively, affects the properties of spheroids. ...
Biofabricated tissues have found numerous applications in tissue engineering and regenerative medicine in addition to the promotion of disease modeling and drug development and screening. Although three-dimensional (3D) printing strategies for designing and developing customized tissue constructs have made significant progress, the complexity of innate multicellular tissues hinders the accurate evaluation of physiological responses in vitro. Cellular aggregates, such as spheroids, are 3D structures where multiple types of cells are co-cultured and organized with endogenously secreted extracellular matrix and are designed to recapitulate the key features of native tissues more realistically. 3D Bioprinting has emerged as a crucial tool for positioning of these spheroids to assemble and organize them into physiologically- and histologically-relevant tissues, mimicking their native counterparts. This has triggered the convergence of spheroid fabrication and bioprinting, leading to the investigation of novel engineering methods for successful assembly of spheroids while simultaneously enhancing tissue repair. This review provides an overview of the current state-of-the-art in spheroid bioprinting methods and elucidates the involved technologies, intensively discusses the recent tissue fabrication applications, outlines the crucial properties that influence the bioprinting of these spheroids and bioprinted tissue characteristics, and finally details the current challenges and future perspectives of spheroid bioprinting efforts in the growing field of biofabrication.
... [80c] In order to tackle the limited oxygen and nutrient diffusion to the core region of tissue strands with diameters above 500 µm, Wu et al. investigated microporous tissue strands by adding alginate microbeads to the cellular suspension prior to injection into the hollow alginate capsules. [88] It is the first time that a porous architecture in the absence of a scaffolding material that has the potential to provide space for blood vessel ingrowth, has been described. The high interconnectivity and porosity of 25% after de-crosslinking resulted in an improved long-term in vitro viability and up-regulation of chondrogenic and osteogenic functionalities. ...
The regeneration and repair of complex structures, interfaces, and mechanical properties present in natural tissues remains a challenge. To move beyond simplified tissue engineered constructs, nature is a source of inspiration for complex, hierarchical scaffold designs. Recent advances in additive manufacturing allow for increasingly complex fabrication of architectures that better mimic the multiscale structure‐function relationship found in natural tissues. In this review, scaffold‐based and scaffold‐free approaches and the synergistic use of fabrication technologies (two things make a third) to produce more biomimetic implants are described. Recent advanced scaffold designs such as auxetic mechanical metamaterials and induced fibrillar alignment are highlighted. Next, the pre‐programmed assembly of spheroids, tissue strands, and other modular building blocks without the need for permanent exogenous scaffold support are discussed. Furthermore, the application of hybridized manufacturing processes to fabricate hierarchical functional constructs is outlined for the osteochondral unit, vascular grafts, and peripheral nerves.
... Micro-structures are essential characteristics of the extracellular matrix that promote nutrient and waste transfer from embedded cells [70]. To investigate the micro-structure of the developed materials, cryo-SEM was used to image the scaffold (Fig. 3). ...
The demands of tissue engineering and regenerative medicine require biomaterials to be accurately deposited into biomimetic shapes, support cellular behaviour and lead to functional tissue formation. Bioinspired yet synthetic biomaterials offer significant advantages over processed, animal-derived products; including high reproducibility and clinical compliance and specific engineered biomimicry of architecture and biological function. Self-assembling peptides are synthetic highly hydrated scaffolds that are rationally designed to mimic the extracellular matrix of a target tissue. Due to the potential benefits of chemically synthesised self-assembling peptides for clinical translation, their development into tools for biofabrication is warranted. However, these systems can be poorly suited to the demands of biofabrication, particularly when functionalised toward tissue-specific conditions. Here, we demonstrate how to improve biofabrication of self-assembling peptides. The fibrillar network arising from the self-assembling peptide Fmoc-FRGDF (containing cell attachment motif RGD) is combined with the robust polysaccharides agarose and alginate demonstrating enhanced printability and cellular compatibility. This study provides a robust methodology for the on-demand printing of personalised implants with a clinically relevant material.
... Few studies have employed the use of coaxial bioprinting to generate capsules for growing tissues in a scaffold-free manner such as cartilage tissue and human adipose-derived stem cells based porous tissue strands. These studies have shown increased cell viability and proliferation compared to solid tissue strands [81,82]. Using this approach, heterocellular tissue strands of rat dermal fibroblasts (RDFs) and beta-TC-3 insulinoma cells were fabricated, which were stained positive to insulin [83] (figures 6(a)-(d)). ...
In the last decade, bioprinting has emerged as a facile technique for fabricating tissues constructs mimicking the architectural complexity and compositional heterogeneity of native tissues. Amongst different bioprinting modalities, extrusion-based bioprinting (EBB) is the most widely used technique. Coaxial bioprinting, a type of EBB, enables fabrication of concentric cell-material layers and enlarges the scope of EBB to mimic several key aspects of native tissues. Over the period of development of bioprinting, tissue constructs integrated with vascular networks, have been one of the major achievements made possible largely by coaxial bioprinting. In this review, current advancements in biofabrication of constructs with coaxial bioprinting are discussed with a focus on different bioinks that are particularly suitable for this modality. This review also expounds the properties of different bioinks suitable for coaxial bioprinting and then analyses the key achievements made by the application of coaxial bioprinting in tissue engineering, drug delivery and in-vitro disease modelling. The major limitations and future perspectives on the critical factors that will determine the ultimate clinical translation of the versatile technique are also presented to the reader.
... These porous strands showed increased cell viability and proliferation rates compared to solid cell strands while maintaining the capability to fuse into a single tissue. In addition, porous scaffold-free samples seeded with adipocyte-derived stem cells showed high viability and functionality in scaffold-free printing when differentiated into chondrogenic and osteogenic lineages [130] ...
The development of appropriate bioinks is a complex task, dependent on the mechanical and biochemical requirements of the final construct and the type of printer used for fabrication. The two most common tissue printers are micro-extrusion and digital light projection printers. Here we briefly discuss the required characteristics of a bioink for each of these printing processes. However, physical printing is only a short window in the lifespan of a printed construct—the system must support and facilitate cellular development after it is printed. To that end, we provide a broad overview of some of the biological molecules currently used as bioinks. Each molecule has advantages for specific tissues/cells, and potential disadvantages are discussed, along with examples of their current use in the field. Notably, it is stressed that active researchers are trending towards the use of composite bioinks. Utilizing the strengths from multiple materials is highlighted as a key component of bioink development.
... To obtain Human ADSCs, surgically discarded adipose tissues were obtained from patients who underwent an elective adipose tissue removal process at the Pennsylvania State University (Hershey, PA) with patient's consent and approval from the Institutional Review Board (IRB protocol # 00004972). Human ADSCs were isolated using the protocol as we previously described [21,22]. The sorted Human ADSCs were cultured in 50:50 mixture of Dulbecco's modified Eagle medium and Ham's nutrient mixture F-12 (DMEM/F12) (Corning, Manassas, VA) supplemented with 20% fetal bovine serum (FBS) (R&D Systems, Minneapolis, MN), 100 U ml −1 penicillin and 100 µg ml −1 streptomycin (Corning, Manassas, VA) at 37 • C with 5% CO 2 . ...
Engineered bone grafts require a vascular network to supply cells with oxygen, nutrients and remove waste. Using heterotypic mature cells to create these grafts in vivo has resulted in limited cell density, ectopic tissue formation and disorganized tissue. Despite evidence that progenitor cell aggregates, such as progenitor spheroids, are a potential candidate for fabrication of native-like pre-vascularized bone tissue, the factors dictating progenitor co-differentiation to create heterotypic pre-vascularized bone tissue remains poorly understood. In this study, we examined a three-dimensional (3D) heterotypic pre-vascularized bone tissue model, using osteogenic and endotheliogenic progenitor spheroids induced by miR-148b and miR-210 mimic transfection, respectively. Spheroids made of transfected cells were assembled into heterotypic structures to determine the impact on co-differentiation as a function of miRNA mimic treatment group and induction time. Our results demonstrated that miRNAs supported the differentiation in heterotypic structures, and that developing heterotypic structures is determined in part by progenitor maturity, as confirmed by gene and protein markers of osteogenic and endotheliogenic differentiation and the mineralization assay. As a proof of concept, miRNA-transfected spheroids were also bioprinted using aspiration-assisted bioprinting and organized into hollow structures to mimic the Haversian canal. Overall, the presented approach could be useful in fabrication of vascularized bone tissue using spheroids as building blocks.
... The SVF was re-suspended in magnetic activated cell sorting (MACS) buffer and loaded into an AutoMACS Pro cell sorter (Milteyni Biotec) to isolate ADSCs using CD90 microbeads (Milteyni Biotec). This was followed by flow cytometry verification of CD73 and CD90 (BD Biosciences), which are specific markers for mesenchymal stem cells [19]. Sorted ADSCs were cultured in DMEM/F12 supplemented with 20% fetal bovine serum (FBS; Gibco), 1 · penicillin and streptomycin (Mediatech), or complete MSC growth media (Promo-Cell) at 37°C with 5% CO 2 . ...
Diabetes is a pandemic manifested through glucose dysregulation mediated via inadequate insulin secretion by beta cells. A beta cell replacement strategy would transform the treatment paradigm from pharmacologic glucose modulation to a genuine cure. Stem cells have emerged as a potential source for beta cell (β-cell) engineering. The detailed generation of functional β-cells from both embryonic and induced pluripotent stem cells has recently been described. Adult stem cells, including adipose derived, may also offer a therapeutic approach but remain ill-defined. In our study, we performed an in-depth assessment of insulin producing beta cells generated from human adipose, irrespective of donor patient age, gender and health status. Cellular transformation was confirmed using flow cytometry and single cell imaging. Insulin secretion was observed with glucose stimulation and abrogated following palmitate exposure; a common free fatty acid implicated in human beta cell dysfunction. We used next generation sequencing to explore gene expression changes prior to and after differentiation of patient matched samples which revealed more than 5000 genes enriched. Adipose derived beta cells displayed comparable gene expression to native β-cells. Pathway analysis demonstrated relevance to stem cell differentiation and pancreatic developmental processes which are vital to cellular function, structural development and regulation. We conclude that the functions associated with adipose derived beta cells is mediated through relevant changes in the transcriptome which resemble those seen in native β-cell morphogenesis and maturation. Therefore, they may represent a viable option for the clinical translation of stem cell-based therapies in diabetes.
... As such, this database search identified studies on a variety of differentiation pathways. The most common hASC differentiation pathways studied were adipogenic (36.9%) [4,5,, osteogenic (32.0%) [4,, and chondrogenic (25.4%) [27,28,[36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][63][64][65][66][67][68][69][70][71][72][73][74][75][76]. Several articles studied a different type of differentiation, which was designated other (4.1%) [50,[77][78][79][80], while 38.5% of articles reviewed did not study any kind of differentiation ( Figure 3). ...
Human adipose-derived stromal/stem cells (hASC) are widely used for in vitro modeling of physiologically relevant human adipose tissue. These models are useful for the development of tissue constructs for soft tissue regeneration and 3-dimensional (3D) microphysiological systems (MPS) for drug discovery. In this systematic review, we report on the current state of hASC culture and assessment methods for adipose tissue engineering using 3D MPS. Our search efforts resulted in the identification of 184 independent records, of which 27 were determined to be most relevant to the goals of the present review. Our results demonstrate a lack of consensus on methods for hASC culture and assessment for the production of physiologically relevant in vitro models of human adipose tissue. Few studies have assessed the impact of different 3D culture conditions on hASC adipogenesis. Additionally, there has been a limited use of assays for characterizing the functionality of adipose tissue in vitro. Results from this study suggest the need for more standardized culture methods and further analysis on in vitro tissue functionality. These will be necessary to validate the utility of 3D MPS as an in vitro model to reduce, refine, and replace in vivo experiments in the drug discovery regulatory process.
