J. Nanotechnol. Eng. Med. 2015;6(2):020301-020301-2. doi:10.1115/1.4031391.

Additive manufacturing, or commonly known as three-dimensional (3D) printing, is a layer-by-layer manufacturing approach enabling fabrication of highly complex objects made of plastics, ceramics, metals, composites, and other emerging materials. 3D bioprinting is an extension of tissue engineering, as it intends to create de novo tissues and organs combining biomaterials, tissue engineering, and 3D printing. It uses bioadditive manufacturing technologies such as laser-based writing, inkjet-based printing, and extrusion-based deposition to print constructs for generation of engineered tissues, tissue constructs, organ modules, and organs. Bioprinting offers great precision on spatial placement of cells, proteins, genes, drugs, and biologically active nano- and micro-particles to better guide tissue generation and formation. This emerging technology appears to be more promising for advancing tissue engineering toward functional tissue and organ fabrication for transplantation, ultimately mitigating organ shortage, and saving lives. In this regard, exploring novel bioprinting processes and next-generation bioprinter technologies, development of new bioink materials and understanding functional tissue and organ formation is of growing importance. This Special Issue selected seven Research Papers and an Expert View article on recent advances, research, and development in 3D printing and bioprinting technologies for tissue engineering and medicine.

Commentary by Dr. Valentin Fuster

Research Papers

J. Nanotechnol. Eng. Med. 2015;6(2):021001-021001-7. doi:10.1115/1.4031466.

Three-dimensional (3D) printing offers versatile possibilities for adapting the structural parameters of tissue engineering scaffolds. However, it is also essential to develop procedures allowing efficient cell seeding independent of scaffold geometry and pore size. The aim of this study was to establish a method for seeding the scaffolds using photopolymerizable cell-laden hydrogels. The latter facilitates convenient preparation, and handling of cell suspension, while distributing the hydrogel precursor throughout the pores, before it is cross-linked with light. In addition, encapsulation of living cells within hydrogels can produce constructs with high initial cell loading and intimate cell-matrix contact, similar to that of the natural extra-cellular matrix (ECM). Three dimensional scaffolds were produced from poly(lactic) acid (PLA) by means of fused deposition modeling. A solution of methacrylamide-modified gelatin (Gel-MOD) in cell culture medium containing photoinitiator Li-TPO-L was used as a hydrogel precursor. Being an enzymatically degradable derivative of natural collagen, gelatin-based matrices are biomimetic and potentially support the process of cell-induced remodeling. Preosteoblast cells MC3T3-E1 at a density of 10 × 106 cells per 1 mL were used for testing the seeding procedure and cell proliferation studies. Obtained results indicate that produced constructs support cell survival and proliferation over extended duration of our experiment. The established two-step approach for scaffold seeding with the cells is simple, rapid, and is shown to be highly reproducible. Furthermore, it enables precise control of the initial cell density, while yielding their uniform distribution throughout the scaffold. Such hybrid tissue engineering constructs merge the advantages of rigid 3D printed constructs with the soft hydrogel matrix, potentially mimicking the process of ECM remodeling.

Commentary by Dr. Valentin Fuster
J. Nanotechnol. Eng. Med. 2015;6(2):021002-021002-9. doi:10.1115/1.4031467.

Current cell-culture is largely performed on synthetic two-dimensional (2D) petri dishes or permeable supports such as Boyden chambers, mostly because of their ease of use and established protocols. It is generally accepted that modern cell biology research requires new physiologically relevant three-dimensional (3D) cell culture platform to mimic in vivo cell responses. To that end, we report the design and development of a suspended hydrogel membrane (ShyM) platform using gelatin methacrylate (GelMA) hydrogel. ShyM thickness (0.25–1 mm) and mechanical properties (10–70 kPa) can be varied by controlling the size of the supporting grid and concentration of GelMA prepolymer, respectively. GelMA ShyMs, with dual media exposure, were found to be compatible with both the cell-seeding and the cell-encapsulation approach as tested using murine 10T1/2 cells and demonstrated higher cellular spreading and proliferation as compared to flat GelMA unsuspended control. The utility of ShyM was also demonstrated using a case-study of invasion of cancer cells. ShyMs, similar to Boyden chambers, are compatible with standard well-plates designs and can be printed using commonly available 3D printers. In the future, ShyM can be potentially extended to variety of photosensitive hydrogels and cell types, to develop new in vitro assays to investigate complex cell–cell and cell–extracellular matrix (ECM) interactions.

Commentary by Dr. Valentin Fuster
J. Nanotechnol. Eng. Med. 2015;6(2):021003-021003-8. doi:10.1115/1.4031174.

Scaffolds play an important role in tissue engineering by providing structural framework and a surface for cells to attach, proliferate, and secrete extracellular matrix (ECM). In order to enable efficient tissue formation, delivering sufficient cells into the scaffold three-dimensional (3D) matrix using traditional static and dynamic seeding methods continues to be a critical challenge. In this study, we investigate a new cell delivery approach utilizing deposition of hydrogel-cell encapsulated microspheroids into polycaprolactone (PCL) scaffolds to improve the seeding efficiency. Three-dimensional-bioplotted PCL constructs (0 deg/90 deg lay down, 284 ± 6 μm strand width, and 555 ± 8 μm strand separation) inoculated with MG-63 model bone cells encapsulated within electrostatically generated calcium-alginate microspheroids (Ø 405 ± 13 μm) were evaluated over seven days in static culture. The microspheroids were observed to be uniformly distributed throughout the PCL scaffold cross section. Encapsulated cells remained viable within the constructs over the test interval with the highest proliferation noted at day 4. This study demonstrates the feasibility of the new approach and highlights the role and critical challenges to be addressed to successfully utilize 3D-bioprinting for microencapsulated cell delivery.

Commentary by Dr. Valentin Fuster
J. Nanotechnol. Eng. Med. 2015;6(2):021004-021004-8. doi:10.1115/1.4031173.

In this study, we report the bioprinting of a three-dimensional (3D) heterogeneous conduit structure encapsulating PC12 neural cells. A core–shell-based hybrid construct is fabricated by combining electrospinning, polymer extrusion, and cell-based bioprinting processes to create a multiscale and multimaterial conduit structure. PC12 nerve cells were shown to be printed with high cell viability (>95%) and to proliferate within the rolled construct at a rate consistent with traditional two-dimensional (2D) culture. Light microscopy and scanning electron microscopy (SEM) have also shown encapsulation of cells within the printed alginate gel and an even cell distribution throughout the heterogeneous cellular construct.

Commentary by Dr. Valentin Fuster
J. Nanotechnol. Eng. Med. 2015;6(2):021005-021005-7. doi:10.1115/1.4031230.

We have developed a three-dimensional (3D) bioprinting system capable of multimaterial and multiscale deposition to enable the next generation of “bottom-up” tissue engineering. This area of research resides at the interface of engineering and life sciences. As such, it entails the design and implementation of diverse elements: a novel hydrogel-based bioink, a 3D bioprinter, automation software, and mammalian cell culture. Our bioprinter has three components uniquely combined into a comprehensive tool: syringe pumps connected to a selector valve that allow precise application of up to five different materials with varying viscosities and chemistries, a high velocity/high-precision x–y–z stage to accommodate the most rapid speeds allowable by the printed materials, and temperature control of the bioink reservoirs, lines, and printing environment. Our custom-designed bioprinter is able to print multiple materials (or multiple cell types in the same material) concurrently with various feature sizes (100 μm–1 mm wide; 100 μm–1 cm high). One of these materials is a biocompatible, printable bioink that has been used to test for cell survival within the hydrogel following printing. Hand-printed (HP) controls show that our bioprinter does not adversely affect the viability of the printed cells. Here, we report the design and build of the 3D bioprinter, the optimization of the bioink, and the stability and viability of our printed constructs.

Topics: Hydrogels , Printing
Commentary by Dr. Valentin Fuster
J. Nanotechnol. Eng. Med. 2015;6(2):021006-021006-5. doi:10.1115/1.4031217.

Bioprinting is a technology that allows making complex tissues from the bottom-up. The need to control accurately both the resolution of the printed droplet and the precision of its positioning was reported. Using a bioink with 1 × 108 cells/mL, we present evidence that the laser-assisted bioprinter (LAB) can deposit droplets of functional mesenchymal stem cells with a resolution of 138 ± 28 μm and a precision of 16 ± 13 μm. We demonstrate that this high printing definition is maintained in three dimensions.

Commentary by Dr. Valentin Fuster
J. Nanotechnol. Eng. Med. 2015;6(2):021007-021007-9. doi:10.1115/1.4031231.

Microfluidic platforms offer revolutionary and practical solutions to challenging problems in biology and medicine. Even though traditional micro/nanofabrication technologies expedited the emergence of the microfluidics field, recent advances in advanced additive manufacturing hold significant potential for single-step, stand-alone microfluidic device fabrication. One such technology, which holds a significant promise for next generation microsystem fabrication is three-dimensional (3D) printing. Presently, building 3D printed stand-alone microfluidic devices with fully embedded microchannels for applications in biology and medicine has the following challenges: (i) limitations in achievable design complexity, (ii) need for a wider variety of transparent materials, (iii) limited z-resolution, (iv) absence of extremely smooth surface finish, and (v) limitations in precision fabrication of hollow and void sections with extremely high surface area to volume ratio. We developed a new way to fabricate stand-alone microfluidic devices with integrated manifolds and embedded microchannels by utilizing a 3D printing and laser micromachined lamination based hybrid manufacturing approach. In this new fabrication method, we exploit the minimized fabrication steps enabled by 3D printing, and reduced assembly complexities facilitated by laser micromachined lamination method. The new hybrid fabrication method enables key features for advanced microfluidic system architecture: (i) increased design complexity in 3D, (ii) improved control over microflow behavior in all three directions and in multiple layers, (iii) transverse multilayer flow and precisely integrated flow distribution, and (iv) enhanced transparency for high resolution imaging and analysis. Hybrid manufacturing approaches hold great potential in advancing microfluidic device fabrication in terms of standardization, fast production, and user-independent manufacturing.

Commentary by Dr. Valentin Fuster

Expert View

J. Nanotechnol. Eng. Med. 2015;6(2):024701-024701-6. doi:10.1115/1.4030414.

Bioprinting is an emerging technology to fabricate artificial tissues and organs through additive manufacturing of living cells in a tissues-specific pattern by stacking them layer by layer. Two major approaches have been proposed in the literature: bioprinting cells in a scaffold matrix to support cell proliferation and growth, and bioprinting cells without using a scaffold structure. Despite great progress, particularly in scaffold-based approaches along with recent significant attempts, printing large-scale tissues and organs is still elusive. This paper demonstrates recent significant attempts in scaffold-based and scaffold-free tissue printing approaches, discusses the advantages and limitations of both approaches, and presents a conceptual framework for bioprinting of scale-up tissue by complementing the benefits of these approaches.

Commentary by Dr. Valentin Fuster

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