Bioink properties before, during and after 3D bioprinting

Bioprinting is a process based on additive manufacturing from materials containing living cells. These materials, often referred to as bioink, are based on cytocompatible hydrogel precursor formulations, which gel in a manner compatible with different bioprinting approaches. The bioink properties before, during and after gelation are essential for its printability, comprising such features as achievable structural resolution, shape fidelity and cell survival. However, it is the final properties of the matured bioprinted tissue construct that are crucial for the end application. During tissue formation these properties are influenced by the amount of cells present in the construct, their proliferation, migration and interaction with the material. A calibrated computational framework is able to predict the tissue development and maturation and to optimize the bioprinting input parameters such as the starting material, the initial cell loading and the construct geometry. In this contribution relevant bioink properties are reviewed and discussed on the example of most popular bioprinting approaches. The effect of cells on hydrogel processing and vice versa is highlighted. Furthermore, numerical approaches were reviewed and implemented for depicting the cellular mechanics within the hydrogel as well as for prediction of mechanical properties to achieve the desired hydrogel construct considering cell density, distribution and material-cell interaction

Construction of 3D in vitro models by bioprinting human pluripotent stem cells: Challenges and opportunities

Three-dimensional (3D) printing of biological material, or 3D bioprinting, is a rapidly expanding field with interesting applications in tissue engineering and regenerative medicine. Bioprinters use cells and biocompatible materials as an ink (bioink) to build 3D structures representative of organs and tissues, in a controlled manner and with micrometric resolution. Human embryonic (hESCs) and induced (hiPSCs) pluripotent stem cells are ideally able to provide all cell types found in the human body. A limited, but growing, number of recent reports suggest that cells derived by differentiation of hESCs and hiPSCs can be used as building blocks in bioprinted human 3D models, reproducing the cellular variety and cytoarchitecture of real tissues. In this review we will illustrate these examples, which include hepatic, cardiac, vascular, corneal and cartilage tissues, and discuss challenges and opportunities of bioprinting more demanding cell types, such as neurons, obtained from human pluripotent stem cells

CPCs and ECM. A good mix for cardiac regeneration

ABSTRACT Despite several improvements in term of diagnosis and prevention, ischemic heart disease still represents one of the principal worldwide causes of death. Cardiac progenitor cells (CPCs) based therapy is considered a valid alternative to heart transplant, but several issues concerning the transplanted cells viability, retention and therapeutic effect need to be solved. Tissue engineering, mixing synthetic or natural polymers with injected cells, could represent the way through which overcomes shortages and set up an effective cardiac regenerative therapy. Nowadays, it is well known that cardiac extracellular matrix (ECM) provides structural and functional integrity, affects cardiac function, development and physiologic repair. In this optic, ECM and ECM-like materials represent functional and biocompatible tools with a great potential to serve as natural or nature-mimicking scaffolds in the field of regenerative medicine. The aim of the present work is to provide an overview on the state of the art and recent advantages on CPCs and scaffold-based therapy for heart regeneration

3D bioprinted human cortical neural constructs derived from induced pluripotent stem cells

Bioprinting techniques use bioinks made of biocompatible non-living materials and cells to build 3D constructs in a controlled manner and with micrometric resolution. 3D bioprinted structures representative of several human tissues have been recently produced using cells derived by differentiation of induced pluripotent stem cells (iPSCs). Human iPSCs can be differentiated in a wide range of neurons and glia, providing an ideal tool for modeling the human nervous system. Here we report a neural construct generated by 3D bioprinting of cortical neurons and glial precursors derived from human iPSCs. We show that the extrusion-based printing process does not impair cell viability in the short and long term. Bioprinted cells can be further differentiated within the construct and properly express neuronal and astrocytic markers. Functional analysis of 3D bioprinted cells highlights an early stage of maturation and the establishment of early network activity behaviors. This work lays the basis for generating more complex and faithful 3D models of the human nervous systems by bioprinting neural cells derived from iPSCs

Co-axial wet-spinning in 3D Bioprinting: state of the art and future perspective of microfluidic integration

Nowadays, 3D bioprinting technologies are rapidly emerging in the field of tissue engineering and regenerative medicine as effective tools enabling the fabrication of advanced tissue constructs that can recapitulate in vitro organ/tissue functions. Selecting the best strategy for bioink deposition is often challenging and time consuming process, as bioink properties-in the first instance, rheological and gelation-strongly influence the suitable paradigms for its deposition. In this short review, we critically discuss one of the available approaches used for bioprinting-namely co-axial wet-spinning extrusion. Such a deposition system, in fact, demonstrated to be promising in terms of printing resolution, shape fidelity and versatility when compared to other methods. An overview of the performances of co-axial technology in the deposition of cellularized hydrogel fibres is discussed, highlighting its main features. Furthermore, we show how this approach allows (i) to decouple the printing accuracy from bioink rheological behaviour-thus notably simplifying the development of new bioinks- A nd (ii) to build heterogeneous multi-materials and/or multicellular constructs that can better mimic the native tissues when combined with microfluidic systems. Finally, the ongoing challenges and the future perspectives for the ultimate fabrication of functional constructs for advanced research studies are highlighted. © 2018 IOP Publishing Ltd

Freeform Bioprinting of Liver Encapsulated in Alginate Hydrogels Tissue Constructs for Pharmacokinetic Study

An in vitro model that can be realistically and inexpensively used to predict human response to various drug administration and toxic chemical exposure is needed. By fabricating a microscale 3D physiological tissue construct consisting of an array of channels and tissue-embedded chambers, one can selectively develop various biomimicking mammalian tissues for a number of pharmaceutical applications, for example, experimental pharmaceutical screening for drug efficacy and toxicity along with apprehending the disposition and metabolic profile of a candidate drug. This paper addresses issues relating to the development and implementation of a bioprinting process for freeform fabrication of a 3D cell-encapsulated hydrogel-based tissue construct, the direct integration onto a microfluidic device for pharmacokinetic study, and the underlying engineering science for the fabrication of a 3D microscale tissue chamber as well as its application in pharmacokinetic study. To this end, a prototype 3D microfluidic tissue chamber embedded with liver cells encapsulated within a hydrogel matrix construct is bioprinted as a physiological in vitro model for pharmacokinetic study. The developed fabrication processes are further validated and parameters optimized by assessing cell viability and liver cell phenotype, in which metabolic and synthetic liver functions are quantitated.Mechanical Engineerin

Hybrid bioprinting of chondrogenically induced human mesenchymal stem cell spheroids

To date, the treatment of articular cartilage lesions remains challenging. A promising strategy for the development of new regenerative therapies is hybrid bioprinting, combining the principles of developmental biology, biomaterial science, and 3D bioprinting. In this approach, scaffold-free cartilage microtissues with small diameters are used as building blocks, combined with a photo-crosslinkable hydrogel and subsequently bioprinted. Spheroids of human bone marrow-derived mesenchymal stem cells (hBM-MSC) are created using a high-throughput microwell system and chondrogenic differentiation is induced during 42 days by applying chondrogenic culture medium and low oxygen tension (5%). Stable and homogeneous cartilage spheroids with a mean diameter of 116 +/- 2.80 mu m, which is compatible with bioprinting, were created after 14 days of culture and a glycosaminoglycans (GAG)- and collagen II-positive extracellular matrix (ECM) was observed. Spheroids were able to assemble at random into a macrotissue, driven by developmental biology tissue fusion processes, and after 72 h of culture, a compact macrotissue was formed. In a directed assembly approach, spheroids were assembled with high spatial control using the bio-ink based extrusion bioprinting approach. Therefore, 14-day spheroids were combined with a photo-crosslinkable methacrylamide-modified gelatin (gelMA) as viscous printing medium to ensure shape fidelity of the printed construct. The photo-initiators Irgacure 2959 and Li-TPO-L were evaluated by assessing their effect on bio-ink properties and the chondrogenic phenotype. The encapsulation in gelMA resulted in further chondrogenic maturation observed by an increased production of GAG and a reduction of collagen I. Moreover, the use of Li-TPO-L lead to constructs with lower stiffness which induced a decrease of collagen I and an increase in GAG and collagen II production. After 3D bioprinting, spheroids remained viable and the cartilage phenotype was maintained. Our findings demonstrate that hBM-MSC spheroids are able to differentiate into cartilage microtissues and display a geometry compatible with 3D bioprinting. Furthermore, for hybrid bioprinting of these spheroids, gelMA is a promising material as it exhibits favorable properties in terms of printability and it supports the viability and chondrogenic phenotype of hBM-MSC microtissues. Moreover, it was shown that a lower hydrogel stiffness enhances further chondrogenic maturation after bioprinting

Printing three-dimensional tissue analogues with decellularized extracellular matrix bioink

The ability to print and pattern all the components that make up a tissue (cells and matrix materials) in three dimensions to generate structures similar to tissues is an exciting prospect of bioprinting. However, the majority of the matrix materials used so far for bioprinting cannot represent the complexity of natural extracellular matrix (ECM) and thus are unable to reconstitute the intrinsic cellular morphologies and functions. Here, we develop a method for the bioprinting of cell-laden constructs with novel decellularized extracellular matrix (dECM) bioink capable of providing an optimized microenvironment conducive to the growth of three-dimensional structured tissue. We show the versatility and flexibility of the developed bioprinting process using tissue-specific dECM bioinks, including adipose, cartilage and heart tissues, capable of providing crucial cues for cells engraftment, survival and long-term function. We achieve high cell viability and functionality of the printed dECM structures using our bioprinting method.open11349353sciescopu

Directional control of angiogenesis to produce a designed multiscale micro-vascular network with bioprinting

Department of Biomedical EngineeringThe biomimetic vascular network is a key element in regeneration of viable, functional and scalable artificial tissues. In this study, we developed a multiscale vascular network that can be patterned freely by using bioprinting technology. An endothelialized channel of several hundred micrometer scale was directly printed. The micro-vascular network consisting of tubular structures of several tens of micrometers was generated through the direction control of angiogenic sprouting using the chemotaxis effect. For this purpose, human umbilical vein endothelial cells (HUVEC) and angiogenic factor secreting cells, normal human dermal fibroblasts (NHDF), were co-patterned at 1 to 2 mm intervals using water soluble bio-ink and alginate based bio-ink, respectively. Then, a bridge pattern connecting the two patterned gels was made with fibrin gel. After printing, an endothelialized channel of about 800 ??m was formed by selective removal of water soluble bio-ink. The angiogenic sprouting was induced at about 200 ??m/day along the bridge pattern from the channel. It was also possible to fabricate a multiscale micro-vascular network with diagonal, wave and branch shapes using bridge patterns of various designs. In this study, we investigated the functionality of hepatocytes by co-culturing mouse primary hepatocytes after fabricating a vascular construct with hepatic lobule-shaped pattern to confirm the utility of the constructed process. As a result, we could confirm largely improved albumin and urea secretion. Based on these results, we confirmed that the tissue specific multiscale vascular network could be constructed. This technique should provide a useful tool for the development of functional and scalable vascularized tissues.clos

Nanogels for pharmaceutical and biomedical applications and their fabrication using 3D printing technologies

Nanogels are hydrogels formed by connecting nanoscopic micelles dispersed in an aqueous medium, which give an opportunity for incorporating hydrophilic payloads to the exterior of the micellar networks and hydrophobic payloads in the core of the micelles. Biomedical and pharmaceutical applications of nanogels have been explored for tissue regeneration, wound healing, surgical device, implantation, and peroral, rectal, vaginal, ocular, and transdermal drug delivery. Although it is still in the early stages of development, due to the increasing demands of precise nanogel production to be utilized for personalized medicine, biomedical applications, and specialized drug delivery, 3D printing has been explored in the past few years and is believed to be one of the most precise, efficient, inexpensive, customizable, and convenient manufacturing techniques for nanogel production