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

The advent of three-dimensional (3D) bioprinting has enabled impressive progress in the development of 3D cellular constructs to mimic the structural and functional characteristics of natural tissues. Bioprinting has considerable translational potential in tissue engineering and regenerative medicine. This review highlights the rational design and biofabrication strategies of diverse 3D bioprinted tissue constructs for orthopedic tissue engineering applications. First, we elucidate the fundamentals of 3D bioprinting techniques and biomaterial inks and discuss the basic design principles of bioprinted tissue constructs. Next, we describe the rationale and key considerations in 3D bioprinting of tissues in many different aspects. Thereafter, we outline the recent advances in 3D bioprinting technology for orthopedic tissue engineering applications, along with detailed strategies of the engineering methods and materials used, and discuss the possibilities and limitations of different 3D bioprinted tissue products. Finally, we summarize the current challenges and future directions of 3D bioprinting technology in orthopedic tissue engineering and regenerative medicine. This review not only delineates the representative 3D bioprinting strategies and their tissue engineering applications, but also provides new insights for the clinical translation of 3D bioprinted tissues to aid in prompting the future development of orthopedic implants. STATEMENT OF SIGNIFICANCE: 3D bioprinting has driven major innovations in the field of tissue engineering and regenerative medicine; aiming to develop a functional viable tissue construct that provides an alternative regenerative therapy for musculoskeletal tissue regeneration. 3D bioprinting-based biofabrication strategies could open new clinical possibilities for creating equivalent tissue substitutes with the ability to customize them to meet patient demands. In this review, we summarize the significance and recent advances in 3D bioprinting technology and advanced bioinks. We highlight the rationale for biofabrication strategies using 3D bioprinting for orthopedic tissue engineering applications. Furthermore, we offer ample perspective and new insights into the current challenges and future direction of orthopedic bioprinting translation research.

3D cell-printing of gradient multi-tissue interfaces for rotator cuff regeneration.

Owing to the prevalence of rotator cuff (RC) injuries and suboptimal healing outcome, rapid and functional regeneration of the tendon-bone interface (TBI) after RC repair continues to be a major clinical challenge. Given the essential role of the RC in shoulder movement, the engineering of biomimetic multi-tissue constructs presents an opportunity for complex TBI reconstruction after RC repair. Here, we propose a gradient cell-laden multi-tissue construct combined with compositional gradient TBI-specific bioinks via 3D cell-printing technology. In vitro studies demonstrated the capability of a gradient scaffold system in zone-specific inducibility and multi-tissue formation mimicking TBI. The regenerative performance of the gradient scaffold on RC regeneration was determined using a rat RC repair model. In particular, we adopted nondestructive, consecutive, and tissue-targeted near-infrared fluorescence imaging to visualize the direct anatomical change and the intricate RC regeneration progression in real time in vivo. Furthermore, the 3D cell-printed implant promotes effective restoration of shoulder locomotion function and accelerates TBI healing in vivo. In summary, this study identifies the therapeutic contribution of cell-printed constructs towards functional RC regeneration, demonstrating the translational potential of biomimetic gradient constructs for the clinical repair of multi-tissue interfaces.

Functional Trachea Reconstruction Using 3D-Bioprinted Native-Like Tissue Architecture Based on Designable Tissue-Specific Bioinks.

Functional segmental trachea reconstruction remains a remarkable challenge in the clinic. To date, functional trachea regeneration with alternant cartilage-fibrous tissue-mimetic structure similar to that of the native trachea relying on the three-dimensional (3D) bioprinting technology has seen very limited breakthrough. This fact is mostly due to the lack of tissue-specific bioinks suitable for both cartilage and vascularized fibrous tissue regeneration, as well as the need for firm interfacial integration between stiff and soft tissues. Here, a novel strategy is developed for 3D bioprinting of cartilage-vascularized fibrous tissue-integrated trachea (CVFIT), utilizing photocrosslinkable tissue-specific bioinks. Both cartilage- and fibrous tissue-specific bioinks created by this study provide suitable printability, favorable biocompatibility, and biomimetic microenvironments for chondrogenesis and vascularized fibrogenesis based on the multicomponent synergistic effect through the hybrid photoinitiated polymerization reaction. As such, the tubular analogs are successfully bioprinted and the ring-to-ring alternant structure is tightly integrated by the enhancement of interfacial bonding through the amidation reaction. The results from both the trachea regeneration and the in situ trachea reconstruction demonstrate the satisfactory tissue-specific regeneration along with realization of mechanical and physiological functions. This study thus illustrates the 3D-bioprinted native tissue-like trachea as a promising alternative for clinical trachea reconstruction.

Mechanically and biologically promoted cell-laden constructs generated using tissue-specific bioinks for tendon/ligament tissue engineering applications.

Tendon and ligament tissues provide stability and mobility crucial for musculoskeletal function, but are particularly prone to injury. Owing to poor innate healing capacity, the regeneration of mature and functional tendon/ligament (T/L) poses a formidable clinical challenge. Advanced bioengineering strategies to develop biomimetic tissue implants are highly desired for the treatment of T/L injuries. Here, we presented a cell-based tissue engineering strategy to generate cell-laden tissue constructs comprising stem cells and tissue-specific bioinks using 3D cell-printing technology. We implemented anin vitropreconditioning approach to guide semi-organized T/L-like formation before thein vivoapplication of cell-printed implants. Duringin vitromaturation, tissue-specific decellularized extracellular matrix-based cellular constructs facilitated long-termin vitroculture with high cell viability and promoted tenogenesis with enhanced cellular/structural anisotropy. Moreover, we demonstrated improved cell survival/retention uponin vivoimplantation of pre-matured constructs in nude mice with de novo tendon formation and improved mechanical strength. Althoughin vivomechanical properties of the cell-printed implants were lower than those of human T/L tissues, the results of this study may have significant implications for future cell-based therapies in tendon and ligament regeneration and translational medicine.

A 3D bioprinted hybrid encapsulation system for delivery of human pluripotent stem cell-derived pancreatic islet-like aggregates.

Islet transplantation is a promising treatment for type 1 diabetes. However, treatment failure can result from loss of functional cells associated with cell dispersion, low viability, and severe immune response. To overcome these limitations, various islet encapsulation approaches have been introduced. Among them, macroencapsulation offers the advantages of delivering and retrieving a large volume of islets in one system. In this study, we developed a hybrid encapsulation system composed of a macroporous polymer capsule with stagger-type membrane and assemblable structure, and a nanoporous decellularized extracellular matrix (dECM) hydrogel containing pancreatic islet-like aggregates using 3D bioprinting technique. The outer part (macroporous polymer capsule) was designed to have an interconnected porous architecture, which allows insulin-producingß-cells encapsulated in the hybrid encapsulation system to maintain their cellular behaviors, including viability, cell proliferation, and insulin-producing function. The inner part (nanoporous dECM hydrogel), composed of the 3D biofabricated pancreatic islet-like aggregates, was simultaneously placed into the macroporous polymer capsule in one step. The developed hybrid encapsulation system exhibited biocompatibilityin vitroandin vivoin terms of M1 macrophage polarization. Furthermore, by controlling the printing parameters, we generated islet-like aggregates, improving cell viability and functionality. Moreover, the 3D bioprinted pancreatic islet-like aggregates exhibited structural maturation and functional enhancement associated with intercellular interaction occurring at theß-cell edges. In addition, we also investigated the therapeutic potential of a hybrid encapsulation system by integrating human pluripotent stem cell-derived insulin-producing cells, which are promising to overcome the donor shortage problem. In summary, these results demonstrated that the 3D bioprinting approach facilitates the fabrication of a hybrid islet encapsulation system with multiple materials and potentially improves the clinical outcomes by driving structural maturation and functional improvement of cells.

Development of 3D Printed Bruch's Membrane-Mimetic Substance for the Maturation of Retinal Pigment Epithelial Cells.

Retinal pigment epithelium (RPE) is a monolayer of the pigmented cells that lies on the thin extracellular matrix called Bruch's membrane. This monolayer is the main component of the outer blood-retinal barrier (BRB), which plays a multifunctional role. Due to their crucial roles, the damage of this epithelium causes a wide range of diseases related to retinal degeneration including age-related macular degeneration, retinitis pigmentosa, and Stargardt disease. Unfortunately, there is presently no cure for these diseases. Clinically implantable RPE for humans is under development, and there is no practical examination platform for drug development. Here, we developed porcine Bruch's membrane-derived bioink (BM-ECM). Compared to conventional laminin, the RPE cells on BM-ECM showed enhanced functionality of RPE. Furthermore, we developed the Bruch's membrane-mimetic substrate (BMS) via the integration of BM-ECM and 3D printing technology, which revealed structure and extracellular matrix components similar to those of natural Bruch's membrane. The developed BMS facilitated the appropriate functions of RPE, including barrier and clearance functions, the secretion of anti-angiogenic growth factors, and enzyme formation for phototransduction. Moreover, it could be used as a basement frame for RPE transplantation. We established BMS using 3D printing technology to grow RPE cells with functions that could be used for an in vitro model and RPE transplantation.