The bioprinting roadmap.

This bioprinting roadmap features salient advances in selected applications of the technique and highlights the status of current developments and challenges, as well as envisioned advances in science and technology, to address the challenges to the young and evolving technique. The topics covered in this roadmap encompass the broad spectrum of bioprinting; from cell expansion and novel bioink development to cell/stem cell printing, from organoid-based tissue organization to bioprinting of human-scale tissue structures, and from building cell/tissue/organ-on-a-chip to biomanufacturing of multicellular engineered living systems. The emerging application of printing-in-space and an overview of bioprinting technologies are also included in this roadmap. Due to the rapid pace of methodological advancements in bioprinting techniques and wide-ranging applications, the direction in which the field should advance is not immediately clear. This bioprinting roadmap addresses this unmet need by providing a comprehensive summary and recommendations useful to experienced researchers and newcomers to the field.

Label free process monitoring of 3D bioprinted engineered constructs via dielectric impedance spectroscopy.

Biofabrication processes can affect biological quality attributes of encapsulated cells within constructs. Currently, assessment of the fabricated constructs is performed offline by subjecting the constructs to destructive assays that require staining and sectioning. This drawback limits the translation of biofabrication processes to industrial practice. In this work, we investigate the dielectric response of viable cells encapsulated in bioprinted 3D hydrogel constructs to an applied alternating electric field as a label-free non-destructive monitoring approach. The relationship between ß-dispersion parameters (permittivity change-Δε, Cole-Cole slope factor-α, critical polarization frequency-f c ) over the frequency spectrum and critical cellular quality attributes are investigated. Results show that alginate constructs containing a higher number of viable cells (human adipose derived stem cells-hASC and osteosarcoma cell line-MG63) were characterized by significantly higher Δε and α (both p < 0.05). When extended to bioprinting, results showed that changes in hASC proliferation and viability in response to changes in critical bioprinting parameters (extrusion pressure, temperature, processing time) significantly affected ∆ε, α, and f c . We also demonstrated monitoring of hASC distribution after bioprinting and changes in proliferation over time across the cross-section of a bioprinted medial knee meniscus construct. The trends in ∆ε over time were in agreement with the alamarBlue assay results for the whole construct, but this measurement approach provided a localized readout on the status of encapsulated cells. The findings of this study support the use of dielectric impedance spectroscopy as a label-free and non-destructive method to characterize the critical quality attributes of bioprinted constructs.

Electrical Cell-Substrate Impedance Spectroscopy Can Monitor Age-Grouped Human Adipose Stem Cell Variability During Osteogenic Differentiation.

Human adipose stem cells (hASCs) are an attractive cell source for bone tissue engineering applications. However, a critical issue to be addressed before widespread hASC clinical translation is the dramatic variability in proliferative capacity and osteogenic potential among hASCs isolated from different donors. The goal of this study was to test our hypothesis that electrical cell-substrate impedance spectroscopy (ECIS) could track complex bioimpedance patterns of hASCs throughout proliferation and osteogenic differentiation to better understand and predict variability among hASC populations. Superlots composed of hASCs from young (aged 24-36 years), middle-aged (aged 48-55 years), and elderly (aged 60-81 years) donors were seeded on gold electrode arrays. Complex impedance measurements were taken throughout proliferation and osteogenic differentiation. During osteogenic differentiation, four impedance phases were identified: increase, primary stabilization, drop phase, and secondary stabilization. Matrix deposition was first observed 48-96 hours after the impedance maximum, indicating, for the first time, that ECIS can identify morphological changes that correspond to late-stage osteogenic differentiation. The impedance maximum was observed at day 10.0 in young, day 6.1 in middle-aged, and day 1.3 in elderly hASCs, suggesting that hASCs from younger donors require a longer time to differentiate than do hASCs from older donors, but young hASCs proliferated more and accreted more calcium long-term. This is the first study to use ECIS to predict osteogenic potential of multiple hASC populations and to show that donor age may temporally control onset of osteogenesis. These findings could be critical for development of patient-specific bone tissue engineering and regenerative medicine therapies. Stem Cells Translational Medicine 2017;6:502-511.

3D-Bioprinting of Polylactic Acid (PLA) Nanofiber-Alginate Hydrogel Bioink Containing Human Adipose-Derived Stem Cells.

Bioinks play a central role in 3D-bioprinting by providing the supporting environment within which encapsulated cells can endure the stresses encountered during the digitally driven fabrication process and continue to mature, proliferate, and eventually form extracellular matrix (ECM). In order to be most effective, it is important that bioprinted constructs recapitulate the native tissue milieu as closely as possible. As such, musculoskeletal soft tissue constructs can benefit from bioinks that mimic their nanofibrous matrix constitution, which is also critical to their function. This study focuses on the development and proof-of-concept assessment of a fibrous bioink composed of alginate hydrogel, polylactic acid nanofibers, and human adipose-derived stem cells (hASC) for bioprinting such tissue constructs. First, hASC proliferation and viability were assessed in 3D-bioplotted strands over 16 days in vitro. Then, a human medial knee meniscus digitally modeled using magnetic resonance images was bioprinted and evaluated over 8 weeks in vitro. Results show that the nanofiber-reinforced bioink allowed higher levels of cell proliferation within bioprinted strands, with a peak at day 7, while still maintaining a vast majority of viable cells at day 16. The cell metabolic activity on day 7 was 28.5% higher in this bioink compared to the bioink without nanofibers. Histology of the bioprinted meniscus at both 4 and 8 weeks showed 54% and 147% higher cell density, respectively, in external versus internal regions of the construct. The presence of collagen and proteoglycans was also noted in areas surrounding the hASC, indicating ECM secretion and chondrogenic differentiation.

Large scale industrialized cell expansion: producing the critical raw material for biofabrication processes.

Cellular biomanufacturing technologies are a critical link to the successful application of cell and scaffold based regenerative therapies, organs-on-chip devices, disease models and any products with living cells contained in them. How do we achieve production level quantities of the key ingredient-'the living cells' for all biofabrication processes, including bioprinting and biopatterning? We review key cell expansion based bioreactor operating principles and how 3D culture will play an important role in achieving production quantities of billions to even trillions of anchorage dependent cells. Furthermore, we highlight some of the challenges in the field of cellular biomanufacturing that must be addressed to achieve desired cellular yields while adhering to the key pillars of good manufacturing practices-safety, purity, stability, potency and identity. Biofabrication technologies are uniquely positioned to provide improved 3D culture surfaces for the industrialized production of living cells.

Manufacturing road map for tissue engineering and regenerative medicine technologies.

The Regenerative Medicine Foundation Annual Conference held on May 6 and 7, 2014, had a vision of assisting with translating tissue engineering and regenerative medicine (TERM)-based technologies closer to the clinic. This vision was achieved by assembling leaders in the field to cover critical areas. Some of these critical areas included regulatory pathways for regenerative medicine therapies, strategic partnerships, coordination of resources, developing standards for the field, government support, priorities for industry, biobanking, and new technologies. The final day of this conference featured focused sessions on manufacturing, during which expert speakers were invited from industry, government, and academia. The speakers identified and accessed roadblocks plaguing the field where improvements in advanced manufacturing offered many solutions. The manufacturing sessions included (a) product development toward commercialization in regenerative medicine, (b) process challenges to scale up manufacturing in regenerative medicine, and (c) infrastructure needs for manufacturing in regenerative medicine. Subsequent to this, industry was invited to participate in a survey to further elucidate the challenges to translation and scale-up. This perspective article will cover the lessons learned from these manufacturing sessions and early results from the survey. We also outline a road map for developing the manufacturing infrastructure, resources, standards, capabilities, education, training, and workforce development to realize the promise of TERM.

Controlled release of metronidazole from composite poly-ε-caprolactone/alginate (PCL/alginate) rings for dental implants.

OBJECTIVE: Dental implants provide support for dental crowns and bridges by serving as abutments for the replacement of missing teeth. To prevent bacterial accumulation and growth at the site of implantation, solutions such as systemic antibiotics and localized delivery of bactericidal agents are often employed. The objective of this study was to demonstrate a novel method of controlled localized delivery of antibacterial agents to an implant site using a biodegradable custom fabricated ring. METHODS: The study involved incorporating a model antibacterial agent (metronidazole) into custom designed poly-ε-caprolactone/alginate (PCL/alginate) composite rings to produce the intended controlled release profile. The rings can be designed to fit around the body of any root form dental implants of various diameters, shapes and sizes. RESULTS: In vitro release studies indicate that pure (100%) alginate rings exhibited an expected burst release of metronidazole in the first few hours, whereas Alginate/PCL composite rings produced a medium burst release followed by a sustained release for a period greater than 4 weeks. By varying the PCL/alginate weight ratios, we have shown that we can control the amount of antibacterial agents released to provide the minimal inhibitory concentration (MIC) needed for adequate protection. The fabricated composite rings have achieved a 50% antibacterial agent release profile over the first 48 h and the remaining amount slowly released over the remainder of the study period. The PCL/alginate agent release characteristic fits the Ritger-Peppas model indicating a diffusion-based mechanism during the 30-day study period. SIGNIFICANCE: The developed system demonstrates a controllable drug release profile and the potential for the ring to inhibit bacterial biofilm growth for the prevention of diseases such as peri-implantitis resulting from bacterial infection at the implant site.

Alginate based 3D hydrogels as an in vitro co-culture model platform for the toxicity screening of new chemical entities.

Prediction of human response to potential therapeutic drugs is through conventional methods of in vitro cell culture assays and expensive in vivo animal testing. Alternatives to animal testing require sophisticated in vitro model systems that must replicate in vivo like function for reliable testing applications. Advancements in biomaterials have enabled the development of three-dimensional (3D) cell encapsulated hydrogels as in vitro drug screening tissue model systems. In this study, we have developed an in vitro platform to enable high density 3D culture of liver cells combined with a monolayer growth of target breast cancer cell line (MCF-7) in a static environment as a representative example of screening drug compounds for hepatotoxicity and drug efficacy. Alginate hydrogels encapsulated with serial cell densities of HepG2 cells (10(5)-10(8) cells/ml) are supported by a porous poly-carbonate disc platform and co-cultured with MCF-7 cells within standard cell culture plates during a 3 day study period. The clearance rates of drug transformation by HepG2 cells are measured using a coumarin based pro-drug. The platform was used to test for HepG2 cytotoxicity 50% (CT(50)) using commercially available drugs which further correlated well with published in vivo LD(50) values. The developed test platform allowed us to evaluate drug dose concentrations to predict hepatotoxicity and its effect on the target cells. The in vitro 3D co-culture platform provides a scalable and flexible approach to test multiple-cell types in a hybrid setting within standard cell culture plates which may open up novel 3D in vitro culture techniques to screen new chemical entity compounds.

Mechanical evaluation of porous titanium (Ti6Al4V) structures with electron beam melting (EBM).

Patient specific porous implants for the reconstruction of craniofacial defects have gained importance due to their better performance over their generic counterparts. The recent introduction of electron beam melting (EBM) for the processing of titanium has led to a one step fabrication of porous custom titanium implants with controlled porosity to meet the requirements of the anatomy and functions at the region of implantation. This paper discusses an image based micro-structural analysis and the mechanical characterization of porous Ti6Al4V structures fabricated using the EBM rapid manufacturing process. SEM studies have indicated the complete melting of the powder material with no evidence of poor inter-layer bonding. Micro-CT scan analysis of the samples indicate well formed titanium struts and fully interconnected pores with porosities varying from 49.75%-70.32%. Compression tests of the samples showed effective stiffness values ranging from 0.57(+/-0.05)-2.92(+/-0.17)GPa and compressive strength values of 7.28(+/-0.93)-163.02(+/-11.98)MPa. For nearly the same porosity values of 49.75% and 50.75%, with a variation in only the strut thickness in the sample sets, the compressive stiffness and strength decreased significantly from 2.92 GPa to 0.57 GPa (80.5% reduction) and 163.02 MPa to 7.28 MPa (93.54 % reduction) respectively. The grain density of the fabricated Ti6Al4V structures was found to be 4.423 g/cm(3) equivalent to that of dense Ti6Al4V parts fabricated using conventional methods. In conclusion, from a mechanical strength viewpoint, we have found that the porous structures produced by the electron beam melting process present a promising rapid manufacturing process for the direct fabrication of customized titanium implants for enabling personalized medicine.

Long-term cultivation of HepG2 liver cells encapsulated in alginate hydrogels: a study of cell viability, morphology and drug metabolism.

In this study, we have evaluated the use of ultra-sterile alginate hydrogels encapsulated with HepG2 liver cells for applications in high throughput drug screening. We have studied the cellular viability and metabolic capacity of the encapsulated cells in two different alginate structures SLM100 (G:M::40:60) and SLG100 (G:M::60:40). We have also developed protocols to characterize the encapsulated cells within the alginate structure using scanning electron microscopy (SEM) and laser scanning confocal microscopy (LSCM). Further we have studied the Phase-I/II metabolic characteristics of the encapsulated cells in monolayer and 3D culture. Our results indicate that cells encapsulated within SLM100 and SLG100 class of alginates have shown high cellular viability with >80% even after 14 days in culture. As expected, the proliferation rates of the encapsulated cells are held steady and do not proliferate within the gels. Production of liver-specific enzymes such as CYP1A1 and CYP3A4 after 14 days in culture indicates the viability and functionality of the encapsulated HepG2 cells. Phase-II Glutathione activity of the encapsulated cells were also maintained in 3D culture conditions. The encapsulated cells within the 3D gels were also capable of metabolizing the pro-drug EFC (7-ethoxy-4-trifluoromethyl coumarin) to HFC (7-hydroxy-4-trifluoromethyl) in a linear fashion over a period of time. These results have provided us with baseline results to benchmark future improvements in material and design configurations for optimal pharmacokinetic response of in vitro tissue model systems.

A Lindenmayer system-based approach for the design of nutrient delivery networks in tissue constructs.

Large thick tissue constructs have reported limited success primarily due to the inability of cells to survive deep within the scaffold. Without access to adequate nutrients, cells placed deep within the tissue construct will die out, leading to non-uniform tissue regeneration. Currently, there is a necessity to design nutrient conduit networks within the tissue construct to enable cells to survive in the matrix. However, the design of complex networks within a tissue construct is challenging. In this paper, we present the Lindenmayer system, an elegant fractal-based language algorithm framework, to generate conduit networks in two- and three-dimensional architecture with several degrees of complexity. The conduit network maintains a parent-child relationship between each branch of the network. Several L-system parameters have been studied-branching angle, branch length, ratio of parent to child branch diameter, etc-to simulate several architectures under a given L-system notation. We have also presented a layered manufacturing-based UV-photopolymerization process using the Texas Instruments DLP system to fabricate the branched structures. This preliminary work showcases the applicability of L-system-based construct designs to drive scaffold fabrication systems.

Enabling sensor technologies for the quantitative evaluation of engineered tissue.

Research in regenerative medicine has necessitated the need for advanced sensing technologies to monitor and evaluate the quality of engineered tissues. Several sensing schemes have been developed to sense specific analytes that enable researchers to assess tissue morphology, growth, and function. In addition to microscopy and staining techniques, tissue engineers are presented with an array of optical, chemical, and biological sensor technologies, which provide them with an opportunity to monitor variables, such as oxygen concentration, pH value, carbon dioxide, and glucose concentration in a noninvasive or minimally invasive manner. The article presents a short description on the core technologies and research reviews on the use of sensors employed in tissue engineering over the past decade. The article concludes by presenting some of the challenges to the further development of these technologies that are capable of real time measurement of tissue structure, composition, and function both for in-vitro and in-vivo analysis.

Application of computer-assisted design in craniofacial reconstructive surgery using a commercial image guidance system. Technical note.

The technology of digital image guidance systems has transformed many aspects of neurosurgery, including intracranial tumor surgery, functional neurosurgery, and spinal surgery. Despite the central role of imaging studies in diagnosis and treatment planning, intraoperative image guidance has so far had very limited application to the surgical correction of craniofacial deformities, particularly those associated with craniosynostosis. The authors report an example of the marriage of computer-assisted design methods to a commercially available neurosurgical image-guidance system in the treatment of a case of anterior plagiocephaly due to unilateral coronal synostosis. They discuss the steps that must yet be taken to make this technology applicable to the management of craniosynostosis in infants.

Computer-aided tissue engineering: application to biomimetic modelling and design of tissue scaffolds.

Computer-aided tissue engineering (CATE) enables many novel approaches in modelling, design and fabrication of complex tissue substitutes with enhanced functionality and improved cell-matrix interactions. Central to CATE is its bio-tissue informatics model that represents tissue biological, biomechanical and biochemical information that serves as a central repository to interface design, simulation and tissue fabrication. The present paper discusses the application of a CATE approach to the biomimetic design of bone tissue scaffold. A general CATE-based process for biomimetic modelling, anatomic reconstruction, computer-assisted-design of tissue scaffold, quantitative-computed-tomography characterization, finite element analysis and freeform extruding deposition for fabrication of scaffold is presented.

Computer-aided tissue engineering: overview, scope and challenges.

Advances in computer-aided technology and its application with biology, engineering and information science to tissue engineering have evolved a new field of computer-aided tissue engineering (CATE). This emerging field encompasses computer-aided design (CAD), image processing, manufacturing and solid free-form fabrication (SFF) for modelling, designing, simulation and manufacturing of biological tissue and organ substitutes. The present Review describes some salient advances in this field, particularly in computer-aided tissue modeling, computer-aided tissue informatics and computer-aided tissue scaffold design and fabrication. Methodologies of development of CATE modelling from high-resolution non-invasive imaging and image-based three-dimensional reconstruction, and various reconstructive techniques for CAD-based tissue modelling generation will be described. The latest development in SFF to tissue engineering and a framework of bio-blueprint modelling for three-dimensional cell and organ printing will also be introduced.