3D Bioprinted Constructs Research Articles

Investigation of cell viability and morphology in 3D bio-printed alginate constructs with tunable stiffness.

In this article, mouse fibroblast cells (L929) were seeded on 2%, 5%, and 10% alginate hydrogels, and they were also bio-printed with 2%, 5%, and 10% alginate solutions individually to form constructs. The elastic and viscous moduli of alginate solutions, their interior structure and stiffness, interactions of cells and alginate, cell viability, migration and morphology were investigated by rheometer, MTT assay, scanning electron microscope (SEM), and fluorescent microscopy. The three types of bio-printed scaffolds of distinctive stiffness were prepared, and the seeded cells showed robust viability either on the alginate hydrogel surfaces or in the 3D bio-printed constructs. Majority of the proliferated cells in the 3D bio-printed constructs weakly attached to the surrounding alginate matrix. The concentration of alginate solution and hydrogel stiffness influenced cell migration and morphology, moreover the cells formed spheroids in the bio-printed 10% alginate hydrogel construct. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1009-1018, 2017.

In Vivo Chondrogenesis in 3D Bioprinted Human Cell-laden Hydrogel Constructs.

Background:The three-dimensional (3D) bioprinting technology allows creation of 3D constructs in a layer-by-layer fashion utilizing biologically relevant materials such as biopolymers and cells. The aim of this study is to investigate the use of 3D bioprinting in a clinically relevant setting to evaluate the potential of this technique for in vivo chondrogenesis.Methods:Thirty-six nude mice (Balb-C, female) received a 5- × 5- × 1-mm piece of bioprinted cell-laden nanofibrillated cellulose/alginate construct in a subcutaneous pocket. Four groups of printed constructs were used: (1) human (male) nasal chondrocytes (hNCs), (2) human (female) bone marrow–derived mesenchymal stem cells (hBMSCs), (3) coculture of hNCs and hBMSCs in a 20/80 ratio, and (4) Cell-free scaffolds (blank). After 14, 30, and 60 days, the scaffolds were harvested for histological, immunohistochemical, and mechanical analysis.Results:The constructs had good mechanical properties and keep their structural integrity after 60 days of implantation. For both the hNC constructs and the cocultured constructs, a gradual increase of glycosaminoglycan production and hNC proliferation was observed. However, the cocultured group showed a more pronounced cell proliferation and enhanced deposition of human collagen II demonstrated by immunohistochemical analysis.Conclusions:In vivo chondrogenesis in a 3D bioprinted human cell-laden hydrogel construct has been demonstrated. The trophic role of the hBMSCs in stimulating hNC proliferation and matrix deposition in the coculture group suggests the potential of 3D bioprinting of human cartilage for future application in reconstructive surgery.

Short-term hypoxic preconditioning promotes prevascularization in 3D bioprinted bone constructs with stromal vascular fraction derived cells.

Reconstruction of complex, craniofacial bone defects often requires autogenous vascularized bone grafts, and still remains a challenge today. In order to address this issue, we isolated the stromal vascular fraction (SVF) from adipose tissues and maintained the phenotypes and the growth of endothelial lineage cells within SVF derived cells (SVFC) by incorporating an endothelial cell medium. We 3D bioprinted SVFC within our hydrogel bioinks and conditioned the constructs in either normoxia or hypoxia. We found that short-term hypoxic conditioning promoted vascularization-related gene expression, whereas long-term hypoxia impaired cell viability and vascularization. 3D bioprinted bone constructs composed of polycaprolactone/hydroxyapatite (PCL/HAp) and SVFC-laden hydrogel bioinks were then implanted into athymic mice, after conditioning in normoxic or short-term hypoxic environments, in order to determine the in vitro and in vivo vascularization and osteogenic differentiation of the constructs. Short-term hypoxic conditioning promoted microvessel formation in vitro and in vivo and promoted integration with existing host vasculature, but did not affect osteogenic differentiation of SVFC. These findings demonstrate the benefit of short-term hypoxia and the potential for utilization of SVFC and 3D bioprinting for generating prevascularized 3D bioprinted bone constructs. Furthermore, the ability to custom design complex anatomical shapes has promising applications for the regeneration of both large and small craniofacial bone defects.

Three-Dimensional Bioprinting and Its Potential in the Field of Articular Cartilage Regeneration.

Three-dimensional (3D) bioprinting techniques can be used for the fabrication of personalized, regenerative constructs for tissue repair. The current article provides insight into the potential and opportunities of 3D bioprinting for the fabrication of cartilage regenerative constructs. Although 3D printing is already used in the orthopedic clinic, the shift toward 3D bioprinting has not yet occurred. We believe that this shift will provide an important step forward in the field of cartilage regeneration. Three-dimensional bioprinting techniques allow incorporation of cells and biological cues during the manufacturing process, to generate biologically active implants. The outer shape of the construct can be personalized based on clinical images of the patient's defect. Additionally, by printing with multiple bio-inks, osteochondral or zonally organized constructs can be generated. Relevant mechanical properties can be obtained by hybrid printing with thermoplastic polymers and hydrogels, as well as by the incorporation of electrospun meshes in hydrogels. Finally, bioprinting techniques contribute to the automation of the implant production process, reducing the infection risk. To prompt the shift from nonliving implants toward living 3D bioprinted cartilage constructs in the clinic, some challenges need to be addressed. The bio-inks and required cartilage construct architecture need to be further optimized. The bio-ink and printing process need to meet the sterility requirements for implantation. Finally, standards are essential to ensure a reproducible quality of the 3D printed constructs. Once these challenges are addressed, 3D bioprinted living articular cartilage implants may find their way into daily clinical practice.

Gelatin methacrylamide hydrogel with graphene nanoplatelets for neural cell-laden 3D bioprinting.

Nervous system is extremely complex which leads to rare regrowth of nerves once injury or disease occurs. Advanced 3D bioprinting strategy, which could simultaneously deposit biocompatible materials, cells and supporting components in a layer-by-layer manner, may be a promising solution to address neural damages. Here we presented a printable nano-bioink composed of gelatin methacrylamide (GelMA), neural stem cells, and bioactive graphene nanoplatelets to target nerve tissue regeneration in the assist of stereolithography based 3D bioprinting technique. We found the resultant GelMA hydrogel has a higher compressive modulus with an increase of GelMA concentration. The porous GelMA hydrogel can provide a biocompatible microenvironment for the survival and growth of neural stem cells. The cells encapsulated in the hydrogel presented good cell viability at the low GelMA concentration. Printed neural construct exhibited well-defined architecture and homogenous cell distribution. In addition, neural stem cells showed neuron differentiation and neurites elongation within the printed construct after two weeks of culture. These findings indicate the 3D bioprinted neural construct has great potential for neural tissue regeneration.

Hydrogel-based reinforcement of 3D bioprinted constructs

Progress within the field of biofabrication is hindered by a lack of suitable hydrogel formulations. Here, we present a novel approach based on a hybrid printing technique to create cellularized 3D printed constructs. The hybrid bioprinting strategy combines a reinforcing gel for mechanical support with a bioink to provide a cytocompatible environment. In comparison with thermoplastics such as -polycaprolactone, the hydrogel-based reinforcing gel platform enables printing at cell-friendly temperatures, targets the bioprinting of softer tissues and allows for improved control over degradation kinetics. We prepared amphiphilic macromonomers based on poloxamer that form hydrolysable, covalently cross-linked polymer networks. Dissolved at a concentration of 28.6%w/w in water, it functions as reinforcing gel, while a 5%w/w gelatin-methacryloyl based gel is utilized as bioink. This strategy allows for the creation of complex structures, where the bioink provides a cytocompatible environment for encapsulated cells. Cell viability of equine chondrocytes encapsulated within printed constructs remained largely unaffected by the printing process. The versatility of the system is further demonstrated by the ability to tune the stiffness of printed constructs between 138 and 263 kPa, as well as to tailor the degradation kinetics of the reinforcing gel from several weeks up to more than a year.

Osteogenic Differentiation of Three-Dimensional Bioprinted Constructs Consisting of Human Adipose-Derived Stem Cells In Vitro and In Vivo.

Here, we aimed to investigate osteogenic differentiation of human adipose-derived stem cells (hASCs) in three-dimensional (3D) bioprinted tissue constructs in vitro and in vivo. A 3D Bio-plotter dispensing system was used for building 3D constructs. Cell viability was determined using live/dead cell staining. After 7 and 14 days of culture, real-time quantitative polymerase chain reaction (PCR) was performed to analyze the expression of osteogenesis-related genes (RUNX2, OSX, and OCN). Western blotting for RUNX2 and immunofluorescent staining for OCN and RUNX2 were also performed. At 8 weeks after surgery, osteoids secreted by osteogenically differentiated cells were assessed by hematoxylin-eosin (H&E) staining, Masson trichrome staining, and OCN immunohistochemical staining. Results from live/dead cell staining showed that most of the cells remained alive, with a cell viability of 89%, on day 1 after printing. In vitro osteogenic induction of the 3D construct showed that the expression levels of RUNX2, OSX, and OCN were significantly increased on days 7 and 14 after printing in cells cultured in osteogenic medium (OM) compared with that in normal proliferation medium (PM). Fluorescence microscopy and western blotting showed that the expression of osteogenesis-related proteins was significantly higher in cells cultured in OM than in cells cultured in PM. In vivo studies demonstrated obvious bone matrix formation in the 3D bioprinted constructs. These results indicated that 3D bioprinted constructs consisting of hASCs had the ability to promote mineralized matrix formation and that hASCs could be used in 3D bioprinted constructs for the repair of large bone tissue defects.

3D bioprinting of human chondrocyte-laden nanocellulose hydrogels for patient-specific auricular cartilage regeneration

Auricular cartilage tissue engineering (TE) aims to provide an effective treatment for patients with acquired or congenital auricular defects. Bioprinting has gained attention in several TE strategies for its ability to spatially control the placement of cells, biomaterials and biological molecules. Although considerable advances have been made to bioprint complex 3D tissue analogues, the development of hydrogel bioinks with good printability and bioactive properties must improve in order to advance the translation of 3D bioprinting into the clinic. In this study, the biological functionality of a bioink composed of nanofibrillated cellulose and alginate (NFC-A) is extensively evaluated for auricular cartilage TE. 3D bioprinted auricular constructs laden with human nasal chondrocytes (hNC) are cultured for up to 28 days and the redifferentiation capacity of hNCs in NFC-A is studied on gene expression as well as on protein levels. 3D bioprinting with NFC-A bioink facilitates the biofabrication of cell-laden, patient-specific auricular constructs with an open inner structure, high cell density and homogenous cell distribution. The cell-laden NFC-A constructs exhibit an excellent shape and size stability as well as an increase in cell viability and proliferation during in vitro culture. Furthermore, NFC-A bioink supports the redifferentiation of hNCs and neo-synthesis of cartilage-specific extracellular matrix components. This demonstrated that NFC-A bioink supports redifferentiation of hNCs while offering proper printability in a biologically relevant aqueous 3D environment, making it a promising tool for auricular cartilage TE and many other biomedical applications.

Prolonged presence of VEGF promotes vascularization in 3D bioprinted scaffolds with defined architecture

Timely vascularization is essential for optimal performance of bone regenerative constructs. Vascularization is efficiently stimulated by vascular endothelial growth factor (VEGF), a substance with a short half-life time. This study investigates the controlled release of VEGF from gelatin microparticles (GMPs) as a means to prolong VEGF activity at the preferred location within 3D bioprinted scaffolds, and the effects on subsequent vascularization. The release of VEGF from GMPs was continuous for 3weeks during in vitro studies, and bioactivity was confirmed using human endothelial progenitor cells (EPCs) in migration assays. Traditional and real-time migration assays showed immediate and efficient EPC migration in the presence of GMP-released VEGF, indistinguishable from VEGF-solution that was added to the medium. Matrigel scaffolds containing EPCs and VEGF, which was released either in a fast or sustained fashion by application of GMPs, were investigated for their in vivo vasculogenic capacity. Implantation in subcutaneous pockets in nude mice for one week demonstrated that vessel formation was significantly higher in the VEGF sustained-release group compared to the fast release group. In addition, regional differences with respect to VEGF release were introduced in 3D bioprinted EPC-laden scaffolds and their influence on vasculogenesis was investigated in vivo. The different regions were retained and vessel formation occurred analogous with the results seen in the Matrigel plugs.We conclude that GMPs are suitable to generate sustained release profiles of bioactive VEGF, and that they can be used to create defined differentiation regions in 3D bioprinted heterogeneous constructs, allowing a new generation of smart scaffold design. The prolonged presence of VEGF led to a significant increase in scaffold vascularization when applied in vivo.

Bioprinting of a mechanically enhanced three-dimensional dual cell-laden construct for osteochondral tissue engineering using a multi-head tissue/organ building system

The aim of this study was to build a mechanically enhanced three-dimensional (3D) bioprinted construct containing two different cell types for osteochondral tissue regeneration. Recently, the production of 3D cell-laden structures using various scaffold-free cell printing technologies has opened up new possibilities. However, ideal 3D complex tissues or organs have not yet been printed because gel-state hydrogels have been used as the principal material and are unable to maintain the desired 3D structure due to their poor mechanical strength. In this study, thermoplastic biomaterial polycaprolactone (PCL), which shows relatively high mechanical properties as compared with hydrogel, was used as a framework for enhancing the mechanical stability of the bioprinted construct. Two different alginate solutions were then infused into the previously prepared framework consisting of PCL to create the 3D construct for osteochondral printing. For this work, a multi-head tissue/organ building system (MtoBS), which was particularly designed to dispense thermoplastic biomaterial and hydrogel having completely different rheology properties, was newly developed and used to bioprint osteochondral tissue. It was confirmed that the line width, position and volume control of PCL and alginate solutions were adjustable in the MtoBS. Most importantly, dual cell-laden 3D constructs consisting of osteoblasts and chondrocytes were successfully fabricated. Further, the separately dispensed osteoblasts and chondrocytes not only retained their initial position and viability, but also proliferated up to 7 days after being dispensed.

Hyaluronic Acid and Dextran-Based Semi-IPN Hydrogels as Biomaterials for Bioprinting

Bioprinting is a recent technology in tissue engineering used for the design of porous constructs through layer-by-layer deposition of cell-laden material. This technology would benefit from new biomaterials that can fulfill specific requirements for the fabrication of well-defined 3D constructs, such as the preservation of cell viability and adequate mechanical properties. We evaluated the suitability of a novel semi-interpenetrating network (semi-IPN), based on hyaluronic acid and hydroxyethyl-methacrylate-derivatized dextran (dex-HEMA), to form 3D hydrogel bioprinted constructs. The rheological properties of the solutions allowed proper handling during bioprinting, whereas photopolymerization led to stable constructs of which their mechanical properties matched the wide range of mechanical strengths of natural tissues. Importantly, excellent viability was observed for encapsulated chondrocytes. The results demonstrate the suitability of hyaluronic acid/dex-HEMA semi-IPNs to manufacture bioprinted constructs for tissue engineering.

Biotechnology: on the trail of evolution

3D bioprinting – the fabrication of geometrically complex 3D structures from biocompatible materials containing living cells using additive manufacturing technologies – is a rapidly developing research field with a broad range of potential applications in fundamental research, regenerative medicine and industry. Currently, research into 3D bioprinting is mostly focused on new therapeutic concepts for the treatment of injured or degenerative tissue by fabrication of functional tissue equivalents or disease models, utilizing mammalian cells. However, 3D bioprinting also has an enormous potential in biotechnology. Due to the defined spatial arrangement of biologically active (non-mammalian) cells in a biomaterial matrix, reaction compartments can be designed according to specific needs, or co-cultures of different cell types can be realized in a highly organized manner to exploit cell-cell interactions. Thus, 3D bioprinting technology can enable new biotechnological concepts, for example, by implementing perfusion systems while protecting shear sensitive cells or performing cascaded bioreactions. Here, we review the use of 3D bioprinting to manufacture defined 3D microenvironments for biotechnological applications using bacteria, fungi, microalgae, plant cells and co-cultures of different cell types. We discuss recent approaches to apply 3D bioprinting in biotechnological applications and – as it is a particular challenge – concepts for the real-time monitoring of the physiological state, growth and metabolic activity of the embedded cells in 3D bioprinted constructs. With these insights, we outline new applications of 3D bioprinting in biotechnology, engineered living materials and space research.