3D printable dynamic hydrogel via entangled biomolecular coacervate towards an ECM-like scaffold.

Three-dimensional (3D) bioprinting technology has emerged as a powerful bio- fabrication platform for tissue engineering because of its ability to engineer living cells and bio-material based 3D objects. Diverse bio-inks based on synthetic and natural biomaterials have also been created and successfully used for tissue regeneration at the same time. Over the past few decades, the fields of tissue engineering and regenerative medicine, which aim to develop functioning tissue-constructs replicating native tissue for the repair and/or replacement of damaged tissues or entire organs, have advanced quickly. Traditional tissue engineering methods, which include scaffolds, growth factors, and cells, had less success fabricating complicated 3D structures and regenerating organs in vivo, which made them logistically and financially unworkable for clinical applications. In this regard, 3D bioprinting, which is an extended application of additive manufacturing is now being explored for tissue engineering and regenerative medicine as it involves the top-down approach of building the Layer-by- layer construction of complicated tissue, thereby producing precise geometries due to controlled nature of matter deposition with the help of anatomically accurate 3D models of the tissue generated by computer graphics. In this article, we seek to present a thorough analysis of the 3D bioprinting techniques, including ink-jet printing, extrusion printing, stereolithography, and laser aided bioprinting methods. With the exact control of structure, dynamics, and biological elements—such as cells and extracellular matrix (ECM)—3D bioprinting has a tremendous deal of promise to build very complex constructions.

3D bioprinting in regenerative medicine: From skin to organ engineering.

The promise of regenerative medicine and tissue engineering is founded on the ability to regenerate diseased or damaged tissues and organs into functional tissues and organs or the creation of new tissues and organs altogether. In theory, damaged and diseased tissues and organs can be regenerated or created using different configurations and combinations of extracellular matrix (ECM), cells, and inductive biomolecules. Regenerative medicine and tissue engineering can allow the improvement of patients’ quality of life through availing novel treatment options. The coupling of regenerative medicine and tissue engineering with 3D printing, big data, and computational algorithms is revolutionizing the treatment of patients in a huge way. 3D bioprinting allows the proper placement of cells and ECMs, allowing the recapitulation of native microenvironments of tissues and organs. 3D bioprinting utilizes different bioinks made up of different formulations of ECM/biomaterials, biomolecules, and even cells. The choice of the bioink used during 3D bioprinting is very important as properties such as printability, compatibility, and physical strength influence the final construct printed. The extracellular matrix (ECM) provides both physical and mechanical microenvironment needed by cells to survive and proliferate. Decellularized ECM bioink contains biochemical cues from the original native ECM and also the right proportions of ECM proteins. Different techniques and characterization methods are used to derive bioinks from several tissues and organs and to evaluate their quality. This review discusses the uses of decellularized ECM bioinks and argues that they represent the most biomimetic bioinks available. In addition, we briefly discuss some polymer-based bioinks utilized in 3D bioprinting.

BackgroundCartilage has limited intrinsic healing capacity, motivating the application of stem cells for regenerative therapies. Therapies to treat osteochondral defects using stem cell therapy-based tissue engineering have been developed and...

Application of three-dimensional (3D) bioprinting in anti-cancer therapy

Organoids provide more realistic in vitro models that closely mimic their corresponding tissues or organs. Three-dimensional (3D) bioprinting allows for precise control over cell distribution, arrangement, and the regulation of cell behavior and interactions, offering flexibility and repeatability. By combining 3D bioprinting with organoid culture, optimal extracellular matrix conditions can be created, better replicating the complex interactions between cells and their environment. The integration of 3D bioprinting amplifies the scalability of organoids, facilitating the development of large-scale organ models that significantly enhance research capabilities. This review explores the crucial role of 3D bioprinting in organoid development, emphasizing its contributions to improving organoid structure and functionality. We discuss how this innovative approach supports the creation of well-defined physiological microenvironments, aids in the development of functional organoids, and enables high-throughput cultivation. Furthermore, we highlight the advantages of optimizing bioprinting strategies and bioinks to advance organoid applications. In addition, we examine the importance of refining 3D bioprinting devices to enhance organoid fabrication and application. Finally, we outline strategies for leveraging 3D bioprinting to further advance organoid research and its implications for regenerative medicine and disease modeling.

Biomaterial-based 3D bioprinting strategy for orthopedic tissue engineering.

Chapter 1 - Nanobiomaterials for three-dimensional bioprinting

The significance of three-dimensional (3D) bioprinting in the domain of regenerative medicine and tissue engineering is readily apparent. To create a multi-functional bioinspired structure, 3D bioprinting requires high-performance bioinks. Bio-inks refer to substances that encapsulate viable cells and are employed in the printing procedure to construct 3D objects progressive through successive layers. For a bio-ink to be considered high-performance, it must meet several critical criteria: printability, gelation kinetics, structural integrity, elasticity and strength, cell adhesion and differentiation, mimicking the native ECM, cell viability and proliferation. As an exemplar application, tissue grafting is used to repair and replace severely injured tissues. The primary considerations in this case include compatibility, availability, advanced surgical techniques, and potential complications after the operation. 3D printing has emerged as an advancement in 3D culture for its use as a regenerative medicine approach. Thus, additive technologies such as 3D bioprinting may offer safe, compatible, and fast-healing tissue engineering options. Multiple methods have been developed for hard and soft tissue engineering during the past few decades, however there are many limitations. Despite significant advances in 3D cell culture, 3D printing, and material creation, a gold standard strategy for designing and rebuilding bone, cartilage, skin, and other tissues has not yet been achieved. Owing to its abundance in the human body and its critical role in protecting and supporting human tissues, soft and hard collagen-based bioinks is an attractive proposition for 3D bioprinting. Collagen, offers a good combination of biocompatibility, controllability, and cell loading. Collagen made of triple helical collagen subunit is a protein-based organic polymer present in almost every extracellular matrix of tissues. Collagen-based bioinks, which create bioinspired scaffolds with multiple functionalities and uses them in various applications, is a represent a breakthrough in the regenerative medicine and biomedical engineering fields. This protein can be blended with a variety of polymers and inorganic fillers to improve the physical and biological performance of the scaffolds. To date, there has not been a comprehensive review appraising the existing literature surround the use of collagen-based bioink applications in 'soft' or 'hard' tissue applications. The uses of the target region in soft tissues include the skin, nerve, and cartilage, whereas in the hard tissues, it specifically refers to bone. For soft tissue healing, collagen-based bioinks must meet greater functional criteria, whereas hard tissue restoration requires superior mechanical qualities. Herein, we summarise collagen-based bioink's features and highlight the most essential ones for diverse healing situations. We conclude with the primary challenges and difficulties of using collagen-based bioinks and suggest future research objectives.

The burden of musculoskeletal diseases in the United States.

Advanced micro- and nanofabrication technologies for tissue engineering

Three-dimensional (3D) bioprinting has advantages for constructing artificial skin tissues in replicating the structures and functions of native skin. Although many studies have presented improved effect of printing skin substitutes in wound healing, using hydrogel inks to fabricate 3D bioprinting architectures with complicated structures, mimicking mechanical properties, and appropriate cellular environments is still challenging. Inspired by collagen nanofibers withstanding stress and regulating cell behavior, a patterned nanofibrous film was introduced to the printed hydrogel scaffold to fabricate a composite artificial skin substitute (CASS). The artificial dermis was printed using gelatin-hyaluronan hybrid hydrogels containing human dermal fibroblasts with gradient porosity and integrated with patterned nanofibrous films simultaneously, while the artificial epidermis was formed by seeding human keratinocytes upon the dermis. The collagen-mimicking nanofibrous film effectively improved the tensile strength and fracture resistance of the CASS, making it sewable for firm implantation into skin defects. Meanwhile, the patterned nanofibrous film also provided the biological cues to guide cell behavior. Consequently, CASS could effectively accelerate the regeneration of large-area skin defects in mouse and pig models by promoting re-epithelialization and collagen deposition. This research developed an effective strategy to prepare composite bioprinting architectures for enhancing mechanical property and regulating cell behavior, and CASS could be a promising skin substitute for treating large-area skin defects.

An artificial construct mimicking the intrinsic properties of the natural extracellular matrix in bones has been considered an ideal platform for bone tissue engineering, as it can present an appropriate microenvironment and regulate cell behaviours. In this report, we introduce biodegradable composite scaffolds consisting of polycaprolactone (PCL) and biphasic calcium phosphate (BCP). The scaffolds were fabricated by a salt-leaching process, and the ability of the scaffolds to facilitate osteogenic differentiation was investigated using human mesenchymal stem cells (hMSCs). The scaffolds had an inter-connected porous structure with quadrilateral pores of approximately 200 ∼ 500 μm in width. The mechanical properties of the scaffolds changed as the BCP content was increased in the starting mixture. In the hMSC experiment, although we found that hMSCs adhered to the surface, as well as the inside, of the scaffolds, the incorporated BCP did not increase the proliferation of the hMSCs over 7 days in culture. Interestingly, the alkaline phosphatase (ALP) activity was 4 times higher on the PCL/BCP composite scaffold (0.12 ± 0.03 nmol/min/μg protein) thanon the PCL scaffold (0.03 ± 0.01 nmol/min/μg protein), suggesting that BCP can aid in generating a local environment that promotes bone regeneration. Therefore, a strategy combining polymers and ceramics can be considered a useful platform for bone tissue engineering.

Due to the limited supply of donor organs and the need for new sources of replacement tissue, researchers continue to strive for new ways to engineer partial and whole organ replacements. A key step is to recreate the 3-dimensional (3D) architecture using extracellular matrix (ECM)—with tissue-specific composition—to provide the mechanical and biochemical cues required to promote tissue development. To address the challenge of creating 3D tissue scaffolds, the field has turned toward tissue decellularization as a source for tissue-specific ECM and as a way of obtaining a cell-free scaffold for tissue engineering and regenerative medicine. The general idea is to remove the cellular material to avoid potential cross-species immune response and adverse reactions during implantation. The goal of the decellularization process is to create a tissue-specific ECM scaffold from donor tissue that can be used to promote wound regeneration and possibly begin to create de novo organs. To date, decellularized tissue has been widely used and shown to promote better regeneration for nerve conduits (Hudson et al., 2004; Sarker et al., 2018), improve cellular adhesion and bioactivity (Hussey et al., 2018; Wolf et al., 2012; Zhang et al., 2016), and can be used for recellularization as a way to introduce patient-specific cells into an organ-derived ECM scaffold (Ott et al., 2008). More recently, decellularized tissues and organs have been utilized as an ideal complex ECM biomaterial for advanced 3D tissue engineering (Jung et al., 2016; Pati et al., 2014). Most decellularization processes begin by removing the tissue or organ of interest from a mouse, rat, or other animal, using standard detergents such a SDS and Triton X-100 to slowly remove cells from the tissue via submersion or perfusion (Crapo et al., 2011). Submersion decellularization is limited to thin tissue samples and tends to be inadequate for whole organ decellularization (Crapo et al., 2011). Perfusion decellularization requires intact vasculature to penetrate the internal tissue structure and enables complete tissue decellularization and subsequent recellularization (Crapo et al., 2011). One potential downside of standard perfusion decellularization processes is the requirement for organ dissection and removal from the animal. In this recent study, Taylor et al. have demonstrated a novel whole animal perfusion decellularization approach that can be used to obtain a completely decellularized rat or can be used to isolate organ systems and specific tissues in a way that maintains both the mechanical integrity and tissue-specific ECM composition of the organ (Taylor et al., 2021). This manuscript uniquely leverages the advantages of perfusion decellularization to demonstrate whole body decellularization of a rat. This is an impressive technical feat which highlights the utility in using native vascular networks to perform decellularization and shows that with their approach any solid organ can be decellularized in situ. The downside to this whole animal approach, as mentioned by the authors, is the 7–9 days of perfusion required to achieve translucent organs (Taylor et al., 2021). Additionally, following whole animal decellularization, the organs or organ systems require subsequent removal to confirm decellularization and for use as future scaffolds or ECM biomaterials. This reduces the need for a whole animal decellularization approach, and in some cases could even add unnecessary processing steps and experimental complexity. To reduce decellularization time and achieve organ-specific decellularization, Taylor et al. excised organs and organ systems using the same methods demonstrated for the whole animal (Taylor et al., 2021). Remarkable decellularization was achieved in both neonatal and adult rats for the abdominal and thoracic compartments, kidney, liver, heart lung pair, and even a lower limb. The vascular networks within these organs and organ systems were intact after decellularization and would likely serve as an ideal conduit for recellularization in future experiments (Taylor et al., 2021). In addition to demonstrating broad applicability in their decellularization method for solid organs, Taylor et al. performed a structural, proteomic, and mechanical evaluation of the decellularized ECM (Taylor et al., 2021). This is important for confirmation that their approach does not eliminate the organ-specific proteins, microstructure, and material properties from the tissue when removing the cells. It also highlights the structural complexity that is left behind such as the glomerulus seen in their scanning electron micrographs of a decellularized kidney. By providing a table of residual ECM proteins from their decellularized heart, kidney, and liver, they have both shown the diversity of the ECM proteins remaining in their scaffolds and created a useful resource for the field to reference when determining important ECM protein distinctions between various organs (Taylor et al., 2021). This resource is particularly useful for the field of ECM material development for tissue engineering and regenerative medicine where researchers are often attempting to recreate the most accurate 3D environment to promote cell differentiation, native tissue mechanical strength, and recreate organ physiological function. This report is a useful addition to the growing field of tissue decellularization by highlighting the simplicity and breadth of perfusion decellularization to produce whole animal and isolated organ system scaffolds in a rat that maintains the tissue ECM protein complexity and mechanical properties. As the field of regenerative medicine and tissue engineering continues to move away from 2D toward 3D platforms as a way to recapitulate the mechanical and biochemical complexity of tissue, methods for efficient decellularization are essential. Scaffolds generated via this approach will likely serve as ideal biomimetic 3D environment for recellularization and can be homogenized and acidified to form the base biomaterial for advanced 3D biofabrication techniques such as 3D bioprinting.

As a microenvironment where cells reside, the extracellular matrix (ECM) has a complex network structure and appropriate mechanical properties to provide structural and biochemical support for the surrounding cells. In tissue engineering, the ECM and its derivatives can mitigate foreign body responses by presenting ECM molecules at the interface between materials and tissues. With the widespread application of three-dimensional (3D) bioprinting, the use of the ECM and its derivative bioinks for 3D bioprinting to replicate biomimetic and complex tissue structures has become an innovative and successful strategy in medical fields. In this review, we summarize the significance and recent progress of ECM-based biomaterials in 3D bioprinting. Then, we discuss the most relevant applications of ECM-based biomaterials in 3D bioprinting, such as tissue regeneration and cancer research. Furthermore, we present the status of ECM-based biomaterials in current research and discuss future development prospects.