3D Cell Printing of Tissue/Organ-Mimicking Constructs for Therapeutic and Drug Testing Applications.

The development of artificial tissue/organs with the functional maturity of their native equivalents is one of the long-awaited panaceas for the medical and pharmaceutical industries. Advanced 3D cell-printing technology and various functional bioinks are promising technologies in the field of tissue engineering that have enabled the fabrication of complex 3D living tissue/organs. Various requirements for these tissues, including a complex and large-volume structure, tissue-specific microenvironments, and functional vasculatures, have been addressed to develop engineered tissue/organs with native relevance. Functional tissue/organ constructs have been developed that satisfy such criteria and may facilitate both in vivo replenishment of damaged tissue and the development of reliable in vitro testing platforms for drug development. This review describes key developments in technologies and materials for engineering 3D cell-printed constructs for therapeutic and drug testing applications.

Development of Liver Decellularized Extracellular Matrix Bioink for Three-Dimensional Cell Printing-Based Liver Tissue Engineering.

The liver is an important organ and plays major roles in the human body. Because of the lack of liver donors after liver failure and drug-induced liver injury, much research has focused on developing liver alternatives and liver in vitro models for transplantation and drug screening. Although numerous studies have been conducted, these systems cannot faithfully mimic the complexity of the liver. Recently, three-dimensional (3D) cell printing technology has emerged as one of a number of innovative technologies that may help to overcome this limitation. However, a great deal of work in developing biomaterials optimized for 3D cell printing-based liver tissue engineering remains. Therefore, in this work, we developed a liver decellularized extracellular matrix (dECM) bioink for 3D cell printing applications and evaluated its characteristics. The liver dECM bioink retained the major ECM components of the liver while cellular components were effectively removed and further exhibited suitable and adjustable properties for 3D cell printing. We further studied printing parameters with the liver dECM bioink to verify the versatility and fidelity of the printing process. Stem cell differentiation and HepG2 cell functions in the liver dECM bioink in comparison to those of commercial collagen bioink were also evaluated, and the liver dECM bioink was found to induce stem cell differentiation and enhance HepG2 cell function. Consequently, the results demonstrate that the proposed liver dECM bioink is a promising bioink candidate for 3D cell printing-based liver tissue engineering.

Extracellular matrix-based sticky sealants for scar-free corneal tissue reconstruction.

Regenerative medicine requires both tissue restoration and ease of compliance for clinical application. Considering this, sticky tissue sealants have been shown to have great potentials over surgical suturing and wound treatment. However, tissue sealants currently used pose challenges such as uncontrollable adhesion formation, mechanical mismatch, and lack of tissue restoration. A new sticky sealant based on gelatinized cornea-derived extracellular matrix (GelCodE) with a visible light-activating system is firstly being introduced in this study. De novo tissue regeneration relies on the matrisome in charge of tissue-organization and development within GelCodE while visible light-based photopolymerization with ruthenium/sodium persulfate rapidly induces covalent bonds with the adjacent tissues. The ease of not only in vivo application, biocompatibility, and biointegration, but also exceptional de novo tissue formation is demonstrated in this study. Interestingly, newly regenerated tissues were shown to have normal tissue-like matrices with little scar formation. Hence, this work presents a promising strategy to meet clinical demands for scar-free tissue recovery with superior ease of clinical application.

Maturation and Protection Effect of Retinal Tissue-Derived Bioink for 3D Cell Printing Technology.

Retinal degeneration is a leading cause of incurable vision loss and blindness. The increasing incidence of retinal degeneration has triggered research into the development of in vitro retinal models for drug development and retinal alternatives for transplantation. However, the complex retinal structure and the retinal microenvironment pose serious challenges. Although 3D cell printing technology has been widely used in tissue engineering, including in vitro model development and regeneration medicine, currently available bioinks are insufficient to recapitulate the complex extracellular matrix environment of the retina. Therefore, in this study, we developed a retinal decellularized extracellular matrix (RdECM) from the porcine retina and evaluated its characteristics. The RdECM conserved the ECM components from the native retina without cellular components. Then, we mixed the RdECM with collagen to form a bioink and confirmed its suitability for 3D cell printing. We further studied the effect of the RdECM bioink on the differentiation of Muller cells. The retinal protective effect of the RdECM bioink was confirmed through a retinal degeneration animal model. Thus, we believe that the RdECM bioink is a promising candidate for retinal tissue engineering.

Three-dimensional cell-printing of advanced renal tubular tissue analogue.

Despite significant progress in the development of renal tissue, recapitulation of perfusable complex renal tubular tissue with clinically relevant cellular heterogeneity is still remaining a challenge. In this study, using coaxial 3D cell-printing technique, we present microfluidic hollow tubes to realize tubular/vascular renal parenchyma composed of renal tubular epithelial and endothelial cells, respectively. We developed a functional hybrid bioink that inherits microenvironments for vascularized native kidney tissue with rapidly crosslinkable character to optimize cell functionality and retain the predefined hollow tubular structure. In addition, the novel bioink and 3D coaxial cell-printing technique provided a complex tube with tunable feature of monolayer and bilayer structure across the length of printed tube. Through prototyping a vascularized renal proximal tubule-on-a-chip, we showed its applicability to novel microfluidic renal tissue models. The renal subcapsular transplantation of the hollow tubes showed a long-term graft survival with the therapeutic capability of the tubular constructs in in vivo model of renal disease, which serves their applicability in regenerative medicine.

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.

Directed differential behaviors of multipotent adult stem cells from decellularized tissue/organ extracellular matrix bioinks.

The decellularized tissue/organ extracellular matrix (dECM) is a naturally derived biomaterial that inherits various functional components from the native tissue or organ. Recently, various kinds of tissue/organ dECM bioinks capable of encapsulating cells, combined with 3D cell printing, have enabled remarkable progress in tissue engineering and regenerative medicine. However, the way in which the dECM component compositions of each tissue of different origins interact with cells and dictate tissue-specific cell behavior in the 3D microenvironment remains mostly unknown. To address this issue, in-depth differential proteomic analyses of four porcine dECMs were performed. Specifically, the differential variations of matrisome protein composition in each decellularized tissue type were also uncovered, which can play a significant role by affecting the resident cells in specific tissues. Furthermore, microarray analyses of human bone marrow mesenchymal stem cells (hBMMSCs) printed with various dECM bioinks were conducted to reveal the effect of compositional variations in a tissue-specific manner at the cellular level depending on the multipotency of MSCs. Through whole transcriptome analysis, differential expression patterns of genes were observed in a tissue-specific manner, and this research provides strong evidence of the tissue-specific functionalities of dECM bioinks.

3D cell printing of in vitro stabilized skin model and in vivo pre-vascularized skin patch using tissue-specific extracellular matrix bioink: A step towards advanced skin tissue engineering.

3D cell-printing technique has been under spotlight as an appealing biofabrication platform due to its ability to precisely pattern living cells in pre-defined spatial locations. In skin tissue engineering, a major remaining challenge is to seek for a suitable source of bioink capable of supporting and stimulating printed cells for tissue development. However, current bioinks for skin printing rely on homogeneous biomaterials, which has several shortcomings such as insufficient mechanical properties and recapitulation of microenvironment. In this study, we investigated the capability of skin-derived extracellular matrix (S-dECM) bioink for 3D cell printing-based skin tissue engineering. S-dECM was for the first time formulated as a printable material and retained the major ECM compositions of skin as well as favorable growth factors and cytokines. This bioink was used to print a full thickness 3D human skin model. The matured 3D cell-printed skin tissue using S-dECM bioink was stabilized with minimal shrinkage, whereas the collagen-based skin tissue was significantly contracted during in vitro tissue culture. This physical stabilization and the tissue-specific microenvironment from our bioink improved epidermal organization, dermal ECM secretion, and barrier function. We further used this bioink to print 3D pre-vascularized skin patch able to promote in vivo wound healing. In vivo results revealed that endothelial progenitor cells (EPCs)-laden 3D-printed skin patch together with adipose-derived stem cells (ASCs) accelerates wound closure, re-epithelization, and neovascularization as well as blood flow. We envision that the results of this paper can provide an insightful step towards the next generation source for bioink manufacturing.

The use of the buccal fat pad flap for oral reconstruction.

Many congenital and acquired defects occur in the maxillofacial area. The buccal fat pad flap (BFP) is a simple and reliable flap for the treatment of many of these defects because of its rich blood supply and location, which is close to the location of various intraoral defects. In this article, we have reviewed BFP and the associated anatomical background, surgical techniques, and clinical applications. The surgical procedure is simple and has shown a high success rate in various clinical applications (approximately 90%), including the closure of oroantral fistula, correction of congenital defect, treatment of jaw bone necrosis, and reconstruction of tumor defects. The control of etiologic factors, size of defect, anatomical location of defect, and general condition of patient could influence the prognosis after grafting. In conclusion, BFP is a reliable flap that can be applied to various clinical situations.