3D bioprinting strategy for engineering vascularized tissue models.

Leveraging three-dimensional (3D) bioprinting in the fields of tissue engineering and regenerative medicine has rapidly accelerated progress toward the development of living tissue constructs and biomedical devices. Ongoing vigorous research has pursued the development of 3D in vitro tissue models to replicate the key aspects of human physiology by incorporating relevant cell populations and adequate environmental cues. Given their advantages of being able to intimately mimic the heterogeneity and complexity of their native counterparts, 3D in vitro models hold promise as alternatives to conventional cell cultures or animal models for translational application to model human physiology/pathology and drug screening. Research has highlighted the importance of in vitro models, and a sophisticated biomanufacturing strategy is vitally required. In particular, vascularization is critical for the prolonged survival and functional maturation of the engineered tissues, which has remained one of the major challenges in the establishment of physiologically relevant 3D in vitro models. To this end, 3D bioprinting can efficiently generate solid and reproducible vascularized tissue models with high architectural and compositional similarity to the native tissues, leading to improve the structural maturation and tissue-specific functionality. Multiple bioprinting strategies have been developed to vascularize in vitro tissues by spatially controlled patterning of vascular precursors or generating readily perfusable vascular structures. This review presents an overview of the advanced 3D bioprinting strategies for vascularized tissue model development. We present the key elements for rebuilding functional vasculature in 3D-bioprinted tissue models and discuss the recent achievements in the engineering of 3D vascularized in vitro models using 3D bioprinting. Finally, we delineate the current challenges and future outlooks of 3D bioprinting-based vascularized tissue models.

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.

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 Bioprinting of an In Vitro Model of a Biomimetic Urinary Bladder with a Contract-Release System.

The development of curative therapy for bladder dysfunction is usually hampered owing to the lack of reliable ex vivo human models that can mimic the complexity of the human bladder. To overcome this issue, 3D in vitro model systems offering unique opportunities to engineer realistic human tissues/organs have been developed. However, existing in vitro models still cannot entirely reflect the key structural and physiological characteristics of the native human bladder. In this study, we propose an in vitro model of the urinary bladder that can create 3D biomimetic tissue structures and dynamic microenvironments to replicate the smooth muscle functions of an actual human urinary bladder. In other words, the proposed biomimetic model system, developed using a 3D bioprinting approach, can recreate the physiological motion of the urinary bladder by incorporating decellularized extracellular matrix from the bladder tissue and introducing cyclic mechanical stimuli. The results showed that the developed bladder tissue models exhibited high cell viability and proliferation rate and promoted myogenic differentiation potential given dynamic mechanical cues. We envision the developed in vitro bladder mimicry model can serve as a research platform for fundamental studies on human disease modeling and pharmaceutical testing.

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.

Neural stem cell delivery using brain-derived tissue-specific bioink for recovering from traumatic brain injury.

Traumatic brain injury is one of the leading causes of accidental death and disability. The loss of parts in a severely injured brain induces edema, neuronal apoptosis, and neuroinflammation. Recently, stem cell transplantation demonstrated regenerative efficacy in an injured brain. However, the efficacy of current stem cell therapy needs improvement to resolve issues such as low survival of implanted stem cells and low efficacy of differentiation into respective cells. We developed brain-derived decellularized extracellular matrix (BdECM) bioink that is printable and has native brain-like stiffness. This study aimed to fabricate injured cavity-fit scaffold with BdECM bioink and assessed the utility of BdECM bioink for stem cell delivery to a traumatically injured brain. Our BdECM bioink had shear thinning property for three-dimensional (3D)-cell-printing and physical properties and fiber structures comparable to those of the native brain, which is important for tissue integration after implantation. The human neural stem cells (NSCs) (F3 cells) laden with BdECM bioink were found to be fully differentiated to neurons; the levels of markers for mature differentiated neurons were higher than those observed with collagen bioinkin vitro. Moreover, the BdECM bioink demonstrated potential in defect-fit carrier fabrication with 3D cell-printing, based on the rheological properties and shape fidelity of the material. As F3 cell-laden BdECM bioink was transplanted into the motor cortex of a rat brain, high efficacy of differentiation into mature neurons was observed in the transplanted NSCs; notably increased level of MAP2, a marker of neuronal differentiation, was observed. Furthermore, the transplanted-cell bioink suppressed reactive astrogliosis and microglial activation that may impede regeneration of the injured brain. The brain-specific material reported here is favorable for NSC differentiation and suppression of neuroinflammation and is expected to successfully support regeneration of a traumatically injured brain.

3D cell-printing of biocompatible and functional meniscus constructs using meniscus-derived bioink.

Meniscus injuries are prevalent in orthopedic diagnosis. The reconstruction of the structural inhomogeneity and anisotropy of the meniscus is a major challenge in clinical practice. Meniscal tissue engineering has emerged as a potential alternative for the treatment of various meniscal diseases and injuries. In this study, we developed three-dimensional (3D) cell-printed meniscus constructs using a mixture of polyurethane and polycaprolactone polymers and cell-laden decellularized meniscal extracellular matrix (me-dECM) bioink with high controllability and durable architectural integrity. The me-dECM bioink provided 3D cell-printed meniscus constructs with a conducive biochemical environment that supported growth and promoted the proliferation and differentiation of encapsulated stem cells toward fibrochondrogenic commitment. In addition, we investigated the in vivo performance of the 3D cell-printed meniscus constructs, which exhibited biocompatibility, excellent mechanical properties, and improved biological functionality. These attributes were similar to those of the native meniscus. Collectively, the 3D cell-printing technology and me-dECM bioink facilitate the recapitulation of meniscus tissue specificity in the aspect of the shape and microenvironment for meniscus regeneration. Further, the developed constructs can potentially be applied in clinical practice.

3D cell-printing of tendon-bone interface using tissue-derived extracellular matrix bioinks for chronic rotator cuff repair.

The tendon-bone interface (TBI) in rotator cuffs exhibits a structural and compositional gradient integrated through the fibrocartilaginous transition. Owing to restricted healing capacity, functional regeneration of the TBI is considered a great clinical challenge. Here, we establish a novel therapeutic platform based on 3D cell-printing and tissue-specific bioinks to achieve spatially-graded physiology for functional TBI regeneration. The 3D cell-printed TBI patch constructs are created via a spatial arrangement of cell-laden tendon and bone-specific bioinks in a graded manner, approximating a multi-tissue fibrocartilaginous interface. This TBI patch offers a cell favorable microenvironment, including high cell viability, proliferative capacity, and zonal-specific differentiation of encapsulated stem cells for TBI formationin vitro. Furthermore,in vivoapplication of spatially-graded TBI patches with stem cells demonstrates their regenerative potential, indicating that repair with 3D cell-printed TBI patch significantly accelerates and promotes TBI healing in a rat chronic tear model. Therefore, our findings propose a new therapeutic strategy for functional TBI regeneration using 3D cell-printing and tissue-specific decellularized extracellular matrix bioink-based approach.

Therapeutic effect of decellularized extracellular matrix-based hydrogel for radiation esophagitis by 3D printed esophageal stent.

Radiation esophagitis, the most common acute adverse effect of radiation therapy, leads to unwanted consequences including discomfort, pain, an even death. However, no direct cure exists for patients suffering from this condition, with the harmful effect of ingestion and acid reflux on the damaged esophageal mucosa remaining an unresolved problem. Through the delivery of the hydrogel with stent platform, we aimed to evaluate the regenerative capacity of a tissue-specific decellularized extracellular matrix (dECM) hydrogel on damaged tissues. For this, an esophagus-derived dECM (EdECM) was developed and shown to have superior biofunctionality and rheological properties, as well as physical stability, potentially providing a better microenvironment for tissue development. An EdECM hydrogel-loaded stent was sequentially fabricated using a rotating rod combined 3D printing system that showed structural stability and protected a loaded hydrogel during delivery. Finally, following stent implantation, the therapeutic effect of EdECM was examined in a radiation esophagitis rat model. Our findings demonstrate that EdECM hydrogel delivery via a stent platform can rapidly resolve an inflammatory response, thus promoting a pro-regenerative microenvironment. The results suggest a promising therapeutic strategy for the treatment of radiation esophagitis.

A 3D cell printed muscle construct with tissue-derived bioink for the treatment of volumetric muscle loss.

Volumetric muscle loss (VML) is an irrecoverable injury associated with muscle loss greater than 20%. Although hydrogel-based 3D engineered muscles and the decellularized extracellular matrix (dECM) have been considered for VML treatment, they have shown limited efficacy. We established a novel VML treatment with dECM bioink using 3D cell printing technology. Volumetric muscle constructs composed of cell-laden dECM bioinks were generated with a granule-based printing reservoir. The 3D cell printed muscle constructs exhibited high cell viability without generating hypoxia and enhanced de novo muscle formation in a VML rat model. To improve functional recovery, prevascularized muscle constructs that mimic the hierarchical architecture of vascularized muscles were fabricated through coaxial nozzle printing with muscle and vascular dECM bioinks. Spatially printing tissue-specific dECM bioinks offers organized microenvironmental cues for the differentiation of each cell and improves vascularization, innervation, and functional recovery. Our present results suggest that a 3D cell printing and tissue-derived bioink-based approach could effectively generate biomimetic engineered muscles to improve the treatment of VML injuries.

Three-dimensional printing of a patient-specific engineered nasal cartilage for augmentative rhinoplasty.

Autologous cartilages or synthetic nasal implants have been utilized in augmentative rhinoplasty to reconstruct the nasal shape for therapeutic and cosmetic purposes. Autologous cartilage is considered to be an ideal graft, but has drawbacks, such as limited cartilage source, requirements of additional surgery for obtaining autologous cartilage, and donor site morbidity. In contrast, synthetic nasal implants are abundantly available but have low biocompatibility than the autologous cartilages. Moreover, the currently used nasal cartilage grafts involve additional reshaping processes, by meticulous manual carving during surgery to fit the diverse nose shape of each patient. The final shapes of the manually tailored implants are highly dependent on the surgeons' proficiency and often result in patient dissatisfaction and even undesired separation of the implant. This study describes a new process of rhinoplasty, which integrates three-dimensional printing and tissue engineering approaches. We established a serial procedure based on computer-aided design to generate a three-dimensional model of customized nasal implant, and the model was fabricated through three-dimensional printing. An engineered nasal cartilage implant was generated by injecting cartilage-derived hydrogel containing human adipose-derived stem cells into the implant containing the octahedral interior architecture. We observed remarkable expression levels of chondrogenic markers from the human adipose-derived stem cells grown in the engineered nasal cartilage with the cartilage-derived hydrogel. In addition, the engineered nasal cartilage, which was implanted into mouse subcutaneous region, exhibited maintenance of the exquisite shape and structure, and striking formation of the cartilaginous tissues for 12 weeks. We expect that the developed process, which combines computer-aided design, three-dimensional printing, and tissue-derived hydrogel, would be beneficial in generating implants of other types of tissue.

Cell-printed 3D liver-on-a-chip possessing a liver microenvironment and biliary system.

To overcome the drawbacks of in vitro liver testing during drug development, numerous liver-on-a-chip models have been developed. However, current liver-on-a-chip technologies are labor-intensive, lack extracellular matrix (ECM) essential for liver cells, and lack a biliary system essential for excreting bile acids, which contribute to intestinal digestion but are known to be toxic to hepatocytes. Therefore, fabrication methods for development of liver-on-a-chip models that overcome the above limitations are required. Cell-printing technology enables construction of complex 3D structures with multiple cell types and biomaterials. We used cell-printing to develop a 3D liver-on-a-chip with multiple cell types for co-culture of liver cells, liver decellularized ECM bioink for a 3D microenvironment, and vascular/biliary fluidic channels for creating vascular and biliary systems. A chip with a biliary fluidic channel induced better biliary system creation and liver-specific gene expression and functions compared to a chip without a biliary system. Further, the 3D liver-on-a-chip showed better functionalities than 2D or 3D cultures. The chip was evaluated using acetaminophen and it showed an effective drug response. In summary, our results demonstrate that the 3D liver-on-a-chip we developed is promising in vitro liver test platform for drug discovery.

Development of a radiopaque, long-term drug eluting bioresorbable stent for the femoral-iliac artery.

Tubular tissues exist in various forms purported for blood supply, waste secretion, etc. to aid proper function and maintenance of the human body. Under pathological conditions, however, these tissues may undergo stenosis. A major surgical treatment for stenosis is to implant a medical device called a stent which aims to expand the narrowed tissue and maintain its patency. Most stents are currently made from metals; despite their high mechanical strength, however, interactions with the host tissue often results in restenosis and stent fracture. To solve these problems, a bioresorbable stent (BRS) is proposed as a next generation stent. In this study, a rotating rod combined 3D printing system was developed to fabricate various types of BRSs. In addition, we confirmed that a 1.5 year long-term release of paclitaxel is possible using polymeric materials. Moreover, a stent loaded with contrast powder was fabricated and was successfully viewed under fluoroscopy. The stent was then implanted in the iliac arteries of pigs and no adverse events were observed for up to 8 weeks.

Three-dimensional bioprinting of cell-laden constructs with polycaprolactone protective layers for using various thermoplastic polymers.

Three-dimensional (3D) cell-printed constructs have been recognized as promising biological substitutes for tissue/organ regeneration. They provide tailored physical properties and biological cues via multi-material printing process. In particular, hybrid bioprinting, enabling to use biodegradable synthetic polymers as framework, has been an attractive method to support weak hydrogels. The constructs with controlled architecture and high shape fidelity were fabricated through this method, depositing spatial arrangement of multi-cell types into microscale constructs. Among biodegradable synthetic polymers, polycaprolactone (PCL) has been commonly chosen in fabrication of cell-printed constructs because of its low melting temperature of 60 °C to be dispensed with extrusion-based bioprinting system. However, in addition to PCL, various synthetic polymers have been widely applied for tissue regeneration. These polymers have distinctive characteristics essential for tissue/organ regeneration. Nevertheless, it is difficult to use some polymers, such as poly (lactic-co-glycolic acid) (PLGA) and polylactic acid (PLA) with 3D bioprinting technology because of their high melting temperature to be dispensed, which can result in thermal damage to the cells in the printed constructs during the fabrication process. We present a novel bioprinting method to use various synthetic polymers in fabrication of cell-printed constructs. PCL was introduced as a protective layer to prevent thermal damage caused by high temperature of polymers during fabrication. Remarkable improvement in cellular activities in the printed constructs with PCL layers was observed compared with the construct without PCL. This bioprinting method can be applied to fabricate more tissue-like constructs through the use of various biomaterials.