Transplantation of Bioprinted Tissues and Organs: Technical and Clinical Challenges and Future Perspectives.

: Three-dimensional (3D) bioprinting is a revolutionary technology in building living tissues and organs with precise anatomic control and cellular composition. Despite the great progress in bioprinting research, there has yet to be any clinical translation due to current limitations in building human-scale constructs, which are vascularized and readily implantable. In this article, we review the current limitations and challenges in 3D bioprinting, including in situ techniques, which are one of several clinical translational models to facilitate the application of this technology from bench to bedside. A detailed discussion is made on the technical barriers in the fabrication of scalable constructs that are vascularized, autologous, functional, implantable, cost-effective, and ethically feasible. Clinical considerations for implantable bioprinted tissues are further expounded toward the correction of end-stage organ dysfunction and composite tissue deficits.

Synergistic interplay between human MSCs and HUVECs in 3D spheroids laden in collagen/fibrin hydrogels for bone tissue engineering.

Stem cell encapsulation in hydrogels has been widely employed in tissue engineering, regenerative medicine, organ-on-a-chip devices and gene delivery; however, fabrication of native-like bone tissue using such a strategy has been a challenge, particularly in vitro, due to the limited cell loading densities resulting in weaker cell-cell interactions and lesser extra-cellular matrix deposition. In particular, scalable bone tissue constructs require vascular network to provide enough oxygen and nutrient supplies to encapsulated cells. To enhance stem cell function and generate pre-vascularized network, we here employed collagen/fibrin hydrogel as an encapsulation matrix for the incorporation of human mesenchymal stem cell/human umbilical vein endothelial cell (MSC/HUVEC) spheroids, and investigated their cellular behavior (including cell viability, morphology, proliferation, and gene expression profile) and compared to that of cell suspension- or MSC spheroids-laden hydrogels. MSC/HUVEC spheroids encapsulated in collagen/fibrin hydrogel showed better cell spreading and proliferation, and up-regulated osteogenic differentiation, and demonstrated pre-vascular network formation. Overall, MSC/HUVEC spheroids-laden hydrogels provided a highly suitable 3D microenvironment for bone tissue formation, which can be utilized in various applications, such as but not limited to tissue engineering, disease modeling and drug screening. STATEMENT OF SIGNIFICANCE: Stem cell encapsulation in hydrogels has been widely used in various areas such as tissue engineering, regenerative medicine, organ-on-a-chip devices and gene delivery; however, fabrication of native-like bone tissue using such an approach has been a challenge, particularly in vitro, due to the limited cell loading densities resulting in weaker cell-cell interactions and lesser extra-cellular matrix deposition. Here in this work, we have encapsulated spheroids of human mesenchymal stems cells (MSCs) in collagen/fibrin hydrogel and evaluated their viability, proliferation, osteogenic differentiation, and bone formation potential in vitro with respect to cell suspension-laden hydrogel samples. We have further incorporated human umbilical vein endothelial cells (HUVECs) into MSC spheroids and demonstrated that the presence of HUVECs in 3D spheroid culture in collagen/fibrin gel induced the formation of pre-vascular network, improved cell viability and proliferation, enhanced the osteogenic differentiation of spheroids, and increased their bone mineral deposition. In sum, MSC/HUVEC spheroids laden hydrogels provided a highly suitable 3D microenvironment for bone tissue formation, which can be utilized in various applications, such as but not limited to tissue engineering and regenerative medicine, disease modeling and drug screening.

Sprouting angiogenesis in engineered pseudo islets.

Despite the recent achievements in cell-based therapies for curing type-1 diabetes (T1D), capillarization in beta (ß)-cell clusters is still a major roadblock as it is essential for long-term viability and function of ß-cells in vivo. In this research, we report sprouting angiogenesis in engineered pseudo islets (EPIs) made of mouse insulinoma ßTC3 cells and rat heart microvascular endothelial cells (RHMVECs). Upon culturing in three-dimensional (3D) constructs under angiogenic conditions, EPIs sprouted extensive capillaries into the surrounding matrix. Ultra-morphological analysis through histological sections also revealed presence of capillarization within EPIs. EPIs cultured in 3D constructs maintained their viability and functionality over time while non-vascularized EPIs, without the presence of RHMVECs, could not retain their viability nor functionality. Here we demonstrate angiogenesis in engineered islets, where patient specific stem cell-derived human beta cells can be combined with microvascular endothelial cells in the near future for long-term graft survival in T1D patients.

Porous tissue strands: avascular building blocks for scalable tissue fabrication.

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 human adipose-derived stem cells, and loaded into tubular alginate capsules, followed by de-crosslinking of the capsules. The resultant cellular structure exhibited a porous morphology and formed cell aggregates in the form of strands, called 'porous tissue strands (pTSs).' Three-dimensional reconstructions show that pTSs were able to maintain ∼25% porosity with a high pore interconnectivity (∼85%) for 3 weeks. Owing to the porous structure, pTSs showed up-regulated cell viability and proliferation rate as compared to solid counterparts throughout the culture period. pTSs also demonstrated self-assembly capability through tissue fusion yielding larger-scale patches. In this paper, chondrogenesis and osteogenesis of pTSs were also demonstrated, where the porous microstructure up-regulated both chondrogenic and osteogenic functionalities indicated by cartilage- and bone-specific immunostaining, quantitative biochemical assessment and gene expression. These findings indicated the functionality of pTSs, which possessed controllable porosity and self-assembly capability, and had great potential to be utilized as tissue building blocks in distinct applications such as cartilage and bone regeneration.

Squid Ring Teeth-coated Mesh Improves Abdominal Wall Repair.

BACKGROUND: Hernia repair is a common surgical procedure with polypropylene (PP) mesh being the standard material for correction because of its durability. However, complications such as seroma and pain are common, and repair failures still approach 15% secondary to poor tissue integration. In an effort to enhance mesh integration, we evaluated the applicability of a squid ring teeth (SRT) protein coating for soft-tissue repair in an abdominal wall defect model. SRT is a biologically derived high-strength protein with strong mechanical properties. We assessed tissue integration, strength, and biocompatibility of a SRT-coated PP mesh in a first-time pilot animal study. METHODS: PP mesh was coated with SRT (SRT-PP) and tested for mechanical strength against uncoated PP mesh. Cell proliferation and adhesion studies were performed in vitro using a 3T3 cell line. Rats underwent either PP (n = 3) or SRT-PP (n = 6) bridge mesh implantation in an anterior abdominal wall defect model. Repair was assessed clinically and radiographically, with integration evaluated by histology and mechanical testing at 60 days. RESULTS: Cell proliferation was enhanced on SRT-PP mesh. This was corroborated in vivo by abdominal wall histology, dramatically diminished craniocaudal mesh contraction, improved strength testing, and higher tissue failure strain. There was no increase in seroma or visceral adhesion formation. No foreign body reactions were noted on liver histology. CONCLUSIONS: SRT applied as a coating appears to augment mesh-tissue integration and improve abdominal wall stability following bridged repair. Further studies in larger animals will determine its applicability for hernia repair in patients.

The bioink: A comprehensive review on bioprintable materials.

This paper discusses "bioink", bioprintable materials used in three dimensional (3D) bioprinting processes, where cells and other biologics are deposited in a spatially controlled pattern to fabricate living tissues and organs. It presents the first comprehensive review of existing bioink types including hydrogels, cell aggregates, microcarriers and decellularized matrix components used in extrusion-, droplet- and laser-based bioprinting processes. A detailed comparison of these bioink materials is conducted in terms of supporting bioprinting modalities and bioprintability, cell viability and proliferation, biomimicry, resolution, affordability, scalability, practicality, mechanical and structural integrity, bioprinting and post-bioprinting maturation times, tissue fusion and formation post-implantation, degradation characteristics, commercial availability, immune-compatibility, and application areas. The paper then discusses current limitations of bioink materials and presents the future prospects to the reader.

Current advances and future perspectives in extrusion-based bioprinting.

Extrusion-based bioprinting (EBB) is a rapidly growing technology that has made substantial progress during the last decade. It has great versatility in printing various biologics, including cells, tissues, tissue constructs, organ modules and microfluidic devices, in applications from basic research and pharmaceutics to clinics. Despite the great benefits and flexibility in printing a wide range of bioinks, including tissue spheroids, tissue strands, cell pellets, decellularized matrix components, micro-carriers and cell-laden hydrogels, the technology currently faces several limitations and challenges. These include impediments to organ fabrication, the limited resolution of printed features, the need for advanced bioprinting solutions to transition the technology bench to bedside, the necessity of new bioink development for rapid, safe and sustainable delivery of cells in a biomimetically organized microenvironment, and regulatory concerns to transform the technology into a product. This paper, presenting a first-time comprehensive review of EBB, discusses the current advancements in EBB technology and highlights future directions to transform the technology to generate viable end products for tissue engineering and regenerative medicine.