Showing papers in "Biofabrication in 2019"

Review of alginate-based hydrogel bioprinting for application in tissue engineering.

Abstract: The dawn of 3D printing in medicals has pinned the domain with hopes of vitality in many patients combating with multitude of diseases. Also entitled as Bio-printing, this is appertained to its sequential printing of precursor ink, embodying cells and polymer/composite, in predetermined trajectory. Precursor ink, in addition to cells, constitutes predominantly hydrogels ascribed to its biodegradability and mimic ability of body's anatomy and mechanical features, e.g. bones, etc. This review paper is devoted to explicate bio-printing (3D/4D) of alginate hydrogels, which are the extract from brown algae, through extrusion additive manufacturing. Alginates are salt derivative of alginic acid and constitute long chain of polysaccharides, which furnishes pliability and gelling adeptness to its structure. Alginate hydrogel (employed for extrusion) can be pristine or composite relying on the requisite properties (target application controlled or in-vivo environment) e.g. Alginate-natural (gelatin/agarose/collagen/hyaluronic acid/etc.) and Alginate-synthetic (polyethylene glycol (PEG)/pluronic F127/etc.). Extrusion additive manufacturing of Alginate is preponderate among others with its uncomplicated processing, material efficiency (cut down on wastage), and outspread adaptability for viscosities (0.03-6*104 Pa.s) but the procedure is limited by resolution(200 ?m) in addition to accuracy. However, 3D-fabricated bio-structures display rigidness (unvarying with conditions) that lacks smart response which is reassured by accounting time feature as noteworthy accessory to printing, interpreting as 4D Bio-printing. This review propounds specific processing itinerary for alginate (meanwhile traversing across its composites/blends with natural and synthetic consideration) in extrusion along with its pre-/during/post-processing parameters intrinsic to process. Furthermore, propensity is also presented in its (Alginate extrusion processing) application for tissue engineering, i.e. bones, cartilage (joints), brain (neural), ear, heart (cardiac), eyes (corneal), etc. due to worldwide quandary over accessibility to natural organs for diverse kinds of diseases. Additionally, the review contemplates recently invented advance printing i.e. 4D printing for biotic species with its challenges and future opportunities.

3D printing of complex GelMA-based scaffolds with nanoclay.

Abstract: Photo-crosslinkable gelatin methacrylate (GelMA) has become an attractive ink in 3D printing due to its excellent biological performance. However, limited by low viscosity and long cross-linking time, it is still a challenge to directly print GelMA by extrusion-based 3D printing. Here, to balance the printability and biocompatibility, biomaterial ink composed of GelMA and nanoclay was specially designed. Using this ink, complex scaffolds with high shape fidelity can be easily printed based on the thixotropic property of nanoclay. In this study, we tried to answer some basic printing-required questions of this ink, including the printability window, general properties (porosity, mechanical strength, et al), and biocompatibility. We found that the GelMA/Nanoclay ink enabled printing complex 3D scaffolds, such as a bionic ear and a branched vessel. Furthermore, the addition of nanoclay improved the porosity, increased the mechanical strength, reduced the degradation ratio, and maintained a good biocompatibility of the printed scaffolds. Therefore, this method offers an easy way to print complex scaffolds with good shape fidelity and biological performance, and it might open up new potential applications for the customized therapy of tissue defects.

Osteogenic and angiogenic tissue formation in high fidelity nanocomposite Laponite-gelatin bioinks.

Abstract: Bioprinting of living cells is rapidly developing as an advanced biofabrication approach to engineer tissues. Bioinks can be extruded in three-dimensions (3D) to fabricate complex and hierarchical constructs for implantation. However, a lack of functionality can often be attributed to poor bioink properties. Indeed, advanced bioinks encapsulating living cells should: (i) present optimal rheological properties and retain 3D structure post fabrication, (ii) promote cell viability and support cell differentiation, and (iii) localise proteins of interest (e.g. vascular endothelial growth factor (VEGF)) to stimulate encapsulated cell activity and tissue ingrowth upon implantation. In this study, we present the results of the inclusion of a synthetic nanoclay, Laponite® (LPN) together with a gelatin methacryloyl (GelMA) bioink and the development of a functional cell-instructive bioink. A nanocomposite bioink displaying enhanced shape fidelity retention and interconnected porosity within extrusion-bioprinted fibres was observed. Human bone marrow stromal cell (HBMSC) viability within the nanocomposite showed no significant changes over 21 days of culture in LPN-GelMA (85.60 ± 10.27%), compared to a significant decrease in GelMA from 7 (95.88 ± 2.90%) to 21 days (55.54 ± 14.72%) (p < 0.01). HBMSCs were observed to proliferate in LPN-GelMA with a significant increase in cell number over 21 days (p < 0.0001) compared to GelMA alone. HBMSC-laden LPN-GelMA scaffolds supported osteogenic differentiation evidenced by mineralised nodule formation, including in the absence of the osteogenic drug dexamethasone. Ex vivo implantation in a chick chorioallantoic membrane model, demonstrated excellent integration of the bioink constructs in the vascular chick embryo after 7 days of incubation. VEGF-loaded LPN-GelMA constructs demonstrated significantly higher vessel penetration than GelMA-VEGF (p < 0.0001) scaffolds. Integration and vascularisation was directly related to increased drug absorption and retention by LPN-GelMA compared to LPN-free GelMA. In summary, a novel light-curable nanocomposite bioink for 3D skeletal regeneration supportive of cell growth and growth factor retention and delivery, evidenced by ex vivo vasculogenesis, was developed with potential application in hard and soft tissue reparation.

Cell-printed 3D liver-on-a-chip possessing a liver microenvironment and biliary system.

Abstract: 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.

Wood-based nanocellulose and bioactive glass modified gelatin-alginate bioinks for 3D bioprinting of bone cells.

Abstract: A challenge in the extrusion-based bioprinting is to find a bioink with optimal biological and physicochemical properties. The aim of this study was to evaluate the influence of wood-based cellulose nanofibrils (CNF) and bioactive glass (BaG) on the rheological properties of gelatin-alginate bioinks and the initial responses of bone cells embedded in these inks. CNF modulated the flow behavior of the hydrogels, thus improving their printability. Chemical characterization by SEM-EDX and ion release analysis confirmed the reactivity of the BaG in the hydrogels. The cytocompatibility of the hydrogels was shown to be good, as evidenced by the viability of human osteoblast-like cells (Saos-2) in cast hydrogels. For bioprinting, 4-layer structures were printed from cell-containing gels and crosslinked with CaCl2. Viability, proliferation and alkaline phosphatase activity (ALP) were monitored over 14 d. In the BaG-free gels, Saos-2 cells remained viable, but in the presence of BaG the viability and proliferation decreased in correlation with the increased viscosity. Still, there was a constant increase in the ALP activity in all the hydrogels. Further bioprinting experiments were conducted using human bone marrow-derived mesenchymal stem cells (hBMSCs), a clinically relevant cell type. Interestingly, hBMSCs tolerated the printing process better than Saos-2 cells and the ALP indicated BaG-stimulated early osteogenic commitment. The addition of CNF and BaG to gelatin-alginate bioinks holds great potential for bone tissue engineering applications.

3D bioprinting of hydrogel constructs with cell and material gradients for the regeneration of full-thickness chondral defect using a microfluidic printing head.

Abstract: Osteochondral (OC) tissue is a biphasic material comprised of articular cartilage integrated atop subchondral bone. Damage to this tissue is highly problematic, owing to its intrinsic inability to regenerate functional tissue in response to trauma or disease. Further, the function of the tissue is largely conferred by its compartmentalized zonal microstructure and composition. Current clinical treatments fail to regenerate new tissue that recapitulates this zonal structure. Consequently, regenerated tissue often lacks long-term stability. To address this growing problem, we propose the development of tissue engineered biomaterials that mimic the zonal cartilage organization and extracellular matrix composition through the use of a microfluidic printing head bearing a mixing unit and incorporated into an extrusion-based bioprinter. The system is devised so that multiple bioinks can be delivered either individually or at the same time and rapidly mixed to the extrusion head, and finally deposited through a coaxial nozzle. This enables the deposition of either layers or continuous gradients of chemical, mechanical and biological cues and fabrication of scaffolds with very high shape fidelity and cell viability. Using such a system we bioprinted cell-laden hydrogel constructs recapitulating the layered structure of cartilage, namely, hyaline and calcified cartilage. The construct was assembled out of two bioinks specifically formulated to mimic the extracellular matrices present in the targeted tissues and to ensure the desired biological response of human bone marrow-derived mesenchymal stem cells and human articular chondrocytes. Homogeneous and gradient constructs were thoroughly characterized in vitro with respect to long-term cell viability and expression of hyaline and hypertrophic markers by means of real-time quantitative PCR and immunocytochemical staining. After 21 days of in vitro culture, we observed production of zone-specific matrix. The PCR analysis demonstrated upregulated expression of hypertrophic markers in the homogenous equivalent of calcified cartilage but not in the gradient heterogeneous construct. The regenerative potential was assessed in vivo in a rat model. The histological analysis of surgically damaged rat trochlea revealed beneficial effect of the bioprinted scaffolds on regeneration of OC defect when compared to untreated control.

Shear-induced alignment of collagen fibrils using 3D cell printing for corneal stroma tissue engineering.

Abstract: The microenvironments of tissues or organs are complex architectures comprised of structural proteins including collagen. Particularly, the cornea is organized in a lattice pattern of collagen fibrils which play a significant role in its transparency. This paper introduces a transparent bioengineered corneal structure for transplantation. The structure is fabricated by inducing shear stress to a corneal stroma-derived decellularized extracellular matrix bioink based on a 3D cell printing technique. The printed structure recapitulates the native macrostructure of the cornea with aligned collagen fibrils which results in the construction of a highly matured and transparent cornea stroma analog. The level of shear stress, controlled by the various size of the printing nozzle, manipulates the arrangement of the fibrillar structure. With proper parameter selection, the printed cornea exhibits high cellular alignment capability, indicating a tissue-specific structural organization of collagen fibrils. In addition, this structural regulation enhances critical cellular events in the assembly of collagen over time. Interestingly, the collagen fibrils that remodeled along with the printing path create a lattice pattern similar to the structure of native human cornea after 4 weeks in vivo. Taken together, these results establish the possibilities and versatility of fabricating aligned collagen fibrils; this represents significant advances in corneal tissue engineering.

3D bioprinting of liver spheroids derived from human induced pluripotent stem cells sustain liver function and viability in vitro

Abstract: The liver is responsible for many metabolic, endocrine and exocrine functions. Approximately 2 million deaths per year are associated with liver failure. Modern 3D bioprinting technologies allied with autologous induced pluripotent stem cells (iPS)-derived grafts could represent a relevant tissue engineering approach to treat end stage liver disease patients. However, protocols that accurately recapitulates liver's epithelial parenchyma through bioprinting are still underdeveloped. Here we evaluated the impacts of using single cell dispersion (i.e. obtained from conventional bidimensional differentiation) of iPS-derived parenchymal (i.e. hepatocyte-like cells) versus using iPS-derived hepatocyte-like cells spheroids (i.e. three-dimensional cell culture), both in combination with non-parenchymal cells (e.g. mesenchymal and endothelial cells), into final liver tissue functionality. Single cell constructs showed reduced cell survival and hepatic function and unbalanced protein/amino acid metabolism when compared to spheroid printed constructs after 18 days in culture. In addition, single cell printed constructs revealed epithelial-mesenchymal transition, resulting in rapid loss of hepatocyte phenotype. These results indicates the advantage of using spheroid-based bioprinting, contributing to improve current liver bioprinting technology towards future regenerative medicine applications and liver physiology and disease modeling.

Bioprinting of 3D breast epithelial spheroids for human cancer models.

Abstract: 3D human cancer models provide a better platform for drug efficacy studies than conventional 2D culture, since they recapitulate important aspects of the in vivo microenvironment. While biofabrication has advanced model creation, bioprinting generally involves extruding individual cells in a bioink and then waiting for these cells to self-assemble into a hierarchical 3D tissue. This self-assembly is time consuming and requires complex cellular interactions with other cell types, extracellular matrix components, and growth factors. We therefore investigated if we could directly bioprint pre-formed 3D spheroids in alginate-based bioinks to create a model tissue that could be used almost immediately. Human breast epithelial cell lines were bioprinted as individual cells or as pre-formed spheroids, either in monoculture or co-culture with vascular endothelial cells. While individual breast cells only spontaneously formed spheroids in Matrigel-based bioink, pre-formed breast spheroids maintained their viability, architecture, and function after bioprinting. Bioprinted breast spheroids were more resistant to paclitaxel than individually printed breast cells; however, this effect was abrogated by endothelial cell co-culture. This study shows that 3D cellular structure bioprinting has potential to create tissue models that quickly replicate the tumor microenvironment.

3D bioprinted hydrogel model incorporating β-tricalcium phosphate for calcified cartilage tissue engineering.

Abstract: One promising strategy to reconstruct osteochondral defects relies on 3D bioprinted three-zonal structures comprised of hyaline cartilage, calcified cartilage, and subchondral bone. So far, several studies have pursued the regeneration of either hyaline cartilage or bone in vitro while-despite its key role in the osteochondral region-only few of them have targeted the calcified layer. In this work, we present a 3D biomimetic hydrogel scaffold containing β-tricalcium phosphate (TCP) for engineering calcified cartilage through a co-axial needle system implemented in extrusion-based bioprinting process. After a thorough bioink optimization, we showed that 0.5% w/v TCP is the optimal concentration forming stable scaffolds with high shape fidelity and endowed with biological properties relevant for the development of calcified cartilage. In particular, we investigate the effect induced by ceramic nano-particles over the differentiation capacity of bioprinted bone marrow-derived human mesenchymal stem cells in hydrogel scaffolds cultured up to 21 d in chondrogenic media. To confirm the potential of the presented approach to generate a functional in vitro model of calcified cartilage tissue, we evaluated quantitatively gene expression of relevant chondrogenic (COL1, COL2, COL10A1, ACAN) and osteogenic (ALPL, BGLAP) gene markers by means of RT-qPCR and qualitatively by means of fluorescence immunocytochemistry.

Printability of pulp derived crystal, fibril and blend nanocellulose-alginate bioinks for extrusion 3D bioprinting

Abstract: Background: One of the main challenges for extrusion 3D bioprinting is the identification of non-synthetic bioinks with suitable rheological properties and biocompatibility. Our aim was to optimise and compare the printability of crystal, fibril and blend formulations of novel pulp derived nanocellulose bioinks and assess biocompatibility with human nasoseptal chondrocytes for cartilage bioprinting. Methods: The printability of crystalline, fibrillated and blend formulations of nanocellulose was determined by assessing resolution (grid-line assay), post-printing shape fidelity and rheology (elasticity, viscosity and shear thinning characteristics) and compared these to pure alginate bioinks. The optimised nanocellulose-alginate bioink was bioprinted with human nasoseptal chondrocytes to determine cytotoxicity, metabolic activity and bioprinted construct topography. Results: All nanocellulose-alginate bioink combinations demonstrated a high degree of shear thinning with reversible stress softening behaviour which contributed to post-printing shape fidelity. The unique blend of crystal and fibril nanocellulose bioink exhibited nano- as well as micro-roughness for cellular survival and differentiation, as well as maintaining the most stable construct volume in culture. Human nasoseptal chondrocytes demonstrated high metabolic activity post printing and adopted a rounded chondrogenic phenotype after prolonged culture. Conclusions: This study highlights the favourable rheological, swelling and biocompatibility properties of nanocellulose-alginate bioinks for extrusion-based bioprinting.

Cell-laden four-dimensional bioprinting using near-infrared-triggered shape-morphing alginate/polydopamine bioinks.

Abstract: Four-dimensional (4D) bioprinting of cell-laden constructs with programmable shape-morphing structures has gained increasing attention in the field of biofabrication and tissue engineering. Currently, most of the widely used materials for 4D printing, including N-isopropylacrylamide-based polymers, are not commonly used in bioinks for cell-laden bioprinting. Herein, we propose a facile approach to create cell-laden constructs with near-infrared (NIR)-triggered shape morphing using bioinks based on alginate (the most widely used bioink for cell-laden bioprinting). Three-dimensional (3D) printed bilayered scaffolds with orthogonal structures using concentrated alginate/polydopamine (PDA) inks (14-18 wt%) showed a change in folded shape during NIR-induced dehydration. The deformation angle of the scaffold could be controlled by laser power, irradiation time and the designed patterns of the printed alginate/PDA struts in scaffolds. Then, 3D printed biphasic scaffolds consisting of alginate/PDA and cell-laden hydrogels exhibited programmable shape change under NIR stimulation. Scaffolds were able to maintain their deformed structures, and the printed cells in hydrogels retained high viability during culture in medium for at least 14 days. The biocompatible and commonly used hydrogel bioinks, NIR-triggered shape-morphing structures and maintenance of the deformed shape in the medium give this facile approach great potential for application in the field of 4D bioprinting and 4D biofabrication of artificial tissues and organs.

High density cell seeding affects the rheology and printability of collagen bioinks

Abstract: An advantage of bioprinting is the ability to incorporate cells into the hydrogel bioink allowing for precise control over cell placement within a construct. Previous work found that the printability of collagen bioinks is highly dependent on their rheological properties. The effect of cell density on collagen rheological properties and, therefore, printability has not been assessed. Therefore, the objective of this study was to determine the effects of incorporating cells on the rheology and printability of collagen bioinks. Primary chondrocytes, at densities relevant to cartilage tissue engineering (up to 100 × 106 cells ml-1), were incorporated into 8 mg ml-1 collagen bioinks. Bioink rheological properties before, during, and after gelation as well as printability were assessed. Cell-laden printed constructs were also cultured for up to 14 d to assess longer-term cell behavior. The addition of cells resulted in an increase in the storage modulus and viscosity of the collagen before gelation. However, the storage modulus after gelation and the rate of gelation decreased with increasing cell density. Theoretical models were compared to the rheological data to suggest frameworks that could be used to predict the rheological properties of cell-laden bioinks. Printability testing showed that improved printability could be achieved with higher cell densities. Fourteen-day culture studies showed that the printing process had no adverse effects on the viability or function of printed cells. Overall, this study shows that collagen bioinks are conducive to bioprinting with a wide range of cell densities while maintaining high printability and chondrocyte viability and function.

Microbial transglutaminase induced controlled crosslinking of gelatin methacryloyl to tailor rheological properties for 3D printing.

Abstract: Gelatin methacryloyl (GelMA) is a versatile biomaterial that has been shown to possess many advantages such as good biocompatibility, support for cell growth, tunable mechanical properties, photocurable capability, and low material cost. Due to these superior properties, much research has been carried out to develop GelMA as a bioink for bioprinting. However, there are still many challenges, and one major challenge is the control of its rheological properties to yield good printability. Herein, this study presents a strategy to control the rheology of GelMA through partial enzymatic crosslinking. Unlike other enzymatic crosslinking strategies where the rheological properties could not be controlled once reaction takes place, we could, to a large extent, keep the rheological properties stable by introducing a deactivation step after obtaining the optimized rheological properties. Ca2+-independent microbial transglutaminase (MTGase) was introduced to partially catalyze covalent bond formation between chains of GelMA. The enzyme was then deactivated to prevent further uncontrolled crosslinking that would render the hydrogel not printable. After printing, a secondary post-printing crosslinking step (photo crosslinking) was then introduced to ensure long-term stability of the printed structure for subsequent cell studies. Biocompatibility studies carried out using cells encapsulated in the printed structure showed excellent cell viability for at least 7 d. This strategy for better control of rheological properties of GelMA could more significantly enhance the usability of this material as bioink for bioprinting of cell-laden structures for soft tissue engineering.

A 3D printed PCL/hydrogel construct with zone-specific biochemical composition mimicking that of the meniscus

Abstract: Engineering the meniscus is challenging due to its bizonal structure; the tissue is cartilaginous at the inner portion and fibrous at the outer portion. Here, we constructed an artificial meniscus mimicking the biochemical organization of the native tissue by 3D printing a meniscus shaped PCL scaffold and then impregnating it with agarose (Ag) and gelatin methacrylate (GelMA) hydrogels in the inner and outer regions, respectively. After incubating the constructs loaded with porcine fibrochondrocytes for 8 weeks, we demonstrated that presence of Ag enhanced glycosaminoglycan (GAG) production by about 4 fold (p < 0.001), while GelMA enhanced collagen production by about 50 fold (p < 0.001). In order to mimic the physiological loading environment, meniscus shaped PCL/hydrogel constructs were dynamically stimulated at strain levels gradually increasing from the outer region (2% of initial thickness) towards the inner region (10%). Incorporation of hydrogels protected the cells from the mechanical damage caused by dynamic stress. Dynamic stimulation resulted in increased ratio of collagen type II (COL 2) in the Ag-impregnated inner region (from 50% to 60% of total collagen), and increased ratio of collagen type I (COL 1) in the GelMA-impregnated outer region (from 60% to 70%). We were able to engineer a meniscus, which is cartilage-like at the inner portion and fibrocartilage-like at the outer portion. Our construct has a potential for use as a substitute for total meniscus replacement.

In situ prevascularization designed by laser-assisted bioprinting: effect on bone regeneration.

Abstract: Vascularization plays a crucial role in bone formation and regeneration process. Development of a functional vasculature to improve survival and integration of tissue-engineered bone substitutes remains a major challenge. Biofabrication technologies, such as bioprinting, have been introduced as promising alternatives to overcome issues related to lack of prevascularization and poor organization of vascular networks within the bone substitutes. In this context, this study aimed at organizing endothelial cells in situ, in a mouse calvaria bone defect, to generate a prevascularization with a defined architecture, and promote in vivo bone regeneration. Laser-assisted bioprinting (LAB) was used to pattern Red Fluorescent Protein-labeled endothelial cells into a mouse calvaria bone defect of critical size, filled with collagen containing mesenchymal stem cells and vascular endothelial growth factor. LAB technology allowed safe and controlled in vivo printing of different cell patterns. In situ printing of endothelial cells gave rise to organized microvascular networks into bone defects. At two months, vascularization rate (vr) and bone regeneration rate (br) showed statistically significant differences between the 'random seeding' condition and both 'disc' pattern (vr = +203.6%; br = +294.1%) and 'crossed circle' pattern (vr = +355%; br = +602.1%). These results indicate that in vivo LAB is a valuable tool to introduce in situ prevascularization with a defined configuration and promote bone regeneration.

In Vitro and In Vivo Evaluation of 3D Bioprinted Small-diameter Vasculature with Smooth Muscle and Endothelium

Abstract: The ability to fabricate perfusable, small-diameter vasculature is a foundational step toward generating human tissues/organs for clinical applications. Currently, it is highly challenging to generate vasculature integrated with smooth muscle and endothelium that replicates the complexity and functionality of natural vessels. Here, a novel method for directly printing self-standing, small-diameter vasculature with smooth muscle and endothelium is presented through combining tailored mussel-inspired bioink and unique 'fugitive-migration' tactics, and its effectiveness and advantages over other methods (i.e. traditional alginate/calcium hydrogel, post-perfusion of endothelial cells) are demonstrated. The biologically inspired, catechol-functionalized, gelatin methacrylate (GelMA/C) undergoes rapid oxidative crosslinking in situ to form an elastic hydrogel, which can be engineered with controllable mechanical strength, high cell/tissue adhesion, and excellent bio-functionalization. The results demonstrate the bioprinted vascular construct possessed numerous favorable, biomimetic characteristics such as proper biomechanics, higher tissue affinity, vascularized tissue manufacturing ability, beneficial perfusability and permeability, excellent vasculoactivity, and in vivo autonomous connection (∼2 weeks) as well as vascular remodeling (∼6 weeks). The advanced achievements in creating biomimetic, functional vasculature illustrate significant potential toward generating a complicated vascularized tissue/organ for clinical transplantation.

Process- and bio-inspired hydrogels for 3D bioprinting of soft free-standing neural and glial tissues.

Abstract: A bio-inspired hydrogel for 3D bioprinting of soft free-standing neural tissues is presented. The novel filler-free bioinks were designed by combining natural polymers for extracellular matrix biomimicry with synthetic polymers to endow desirable rheological properties for 3D bioprinting. Crosslinking of thiolated Pluronic F-127 with dopamine-conjugated (DC) gelatin and DC hyaluronic acid through a thiol-catechol reaction resulted in thermally gelling bioinks with Herschel-Bulkley fluid rheological behavior. Microextrusion 3D bioprinting was used to fabricate free-standing cell-laden tissue constructs. The bioinks exhibited flattened parabolic velocity profiles with tunable low shear regions. Two pathways were investigated for curing the bioink: chelation and photocuring. The storage modulus of the cured bioinks ranged from 6.7 to 11.7 kPa. The iron (III) chelation chemistry produced crosslinked neural tissues of relatively lower storage modulus than the photocuring approach. In vitro cell viability studies using the 3D bioprinted neural tissues showed that the cured bioink was biocompatible based on minimal cytotoxic response observed over seven days in culture relative to control studies using alginate hydrogels. Rodent Schwann cell-, rodent neuronal cell-, and human glioma cell-laden tissue constructs were printed and cultured over seven days and exhibited comparable viability relative to alginate bioink controls. The ability to fabricate soft, free-standing 3D neural tissues with low modulus has implications in the biofabrication of microphysiological neural systems for disease modeling as well as neural tissues and innervated tissues for regenerative medicine.

Bioprinters for organs-on-chips.

Abstract: Recent advances in bioprinting technologies have enabled rapid manufacturing of organ-on-chip models along with biomimetic tissue microarchitectures. Bioprinting techniques can be used to integrate microfluidic channels and flow connections in organ-on-chip models. We review bioprinters in two categories of nozzle-based and optical-based methods, and then discuss their fabrication parameters such as resolution, replication fidelity, fabrication time, and cost for micro-tissue models and microfluidic applications. The use of bioprinters has shown successful replicates of functional engineered tissue models integrated within a desired microfluidic system, which facilitates the observation of metabolism or secretion of models and sophisticated control of a dynamic environment. This may provide a wider order of tissue engineering fabrication in mimicking physiological conditions for enhancing further applications such as drug development and pathological studies.

Engineering bioprintable alginate/gelatin composite hydrogels with tunable mechanical and cell adhesive properties to modulate tumor spheroid growth kinetics.

Abstract: Tunable bioprinting materials are capable of creating a broad spectrum of physiological mimicking 3D models enabling in vitro studies that more accurately resemble in vivo conditions. Tailoring the material properties of the bioink such that it achieves both bioprintability and biomimicry remains a key challenge. Here we report the development of engineered composite hydrogels consisting of gelatin and alginate components. The composite gels are demonstrated as a cell-laden bioink to build 3D bioprinted in vitro breast tumor models. The initial mechanical characteristics of each composite hydrogel are correlated to cell proliferation rates and cell spheroid morphology spanning month long culture conditions. MDA-MB-231 breast cancer cells show gel formulation-dependency on the rates and frequency of self-assembly into multicellular tumor spheroids (MCTS). Hydrogel compositions comprised of decreasing alginate concentrations, and increasing gelatin concentrations, result in gels that are mechanically soft and contain a greater number of cell-adhesion moieties driving the development of large MCTS; conversely gels containing increasing alginate, and decreasing gelatin concentrations are mechanically stiffer, with fewer cell-adhesion moieties present in the composite gels yielding smaller and less viable MCTS. These composite hydrogels can be used in the biofabrication of tunable in vitro systems that mimic both the mechanical and biochemical properties of the native tumor stroma.

Collagen/bioceramic-based composite bioink to fabricate a porous 3D hASCs-laden structure for bone tissue regeneration.

Abstract: To successfully achieve the porous cell-blocks, a bioink is a prerequisite requirement. However, although various hydrogel-based bioinks have been applied, a hydrogel/bioceramic-based composite bioink consisting of cells has not been actively investigated owing to its poor printability and low initial cell-viability. In this study, a new bioink consisting of fibrillated collagen, cells, and bioceramic (β-TCP) is suggested to attain a 3D porous cell-laden composite structure with high cellular responses, in aspects of initial cell viability, proliferation, and differentiation using preosteoblasts (MC3T3-E1) and human adipose stem cells (hASCs). By manipulating the processing conditions and weight fractions of the ceramic in the bioink, a 3D porous cell-laden composite structure can be fabricated successfully. The cell-laden composite structure revealed that the printed structure was mechanically stable, the laden cells were satisfactorily viable, and even cell proliferation/differentiation was well performed. Moreover, the cells in the composite structure exhibited significant osteogenic activities compared to the pure collagen bioink (control), and higher levels of osteogenic gene expression of the hASC-laden composite structure were observed without using an osteogenic medium than those of the control using an osteogenic medium, indicating that the laden β-TCP triggered osteogenic differentiation of the hASCs.

Bioprinted osteon-like scaffolds enhance in vivo neovascularization.

Abstract: Bone tissue engineers are facing a daunting challenge when attempting to fabricate bigger constructs intended for use in the treatment of large bone defects, which is the vascularization of the graft. Cell-based approaches and, in particular, the use of in vitro coculture of human umbilical vein endothelial cells (HUVECs) and human mesenchymal stem cells (hMSCs) has been one of the most explored options. We present in this paper an alternative method to mimic the spatial pattern of HUVECs and hMSCs found in native osteons based on the use of extrusion-based 3D bioprinting (3DP). We developed a 3DP biphasic osteon-like scaffold, containing two separate osteogenic and vasculogenic cell populations encapsulated in a fibrin bioink in order to improve neovascularization. To this end, we optimized the fibrin bioink to improve the resolution of printed strands and ensure a reproducible printing process; the influence of printing parameters on extruded strand diameter and cell survival was also investigated. The mechanical strength of the construct was improved by co-printing the fibrin bioink along a supporting PCL carrier scaffold. Compressive mechanical testing showed improved mechanical properties with an average compressive modulus of 131 ± 23 MPa, which falls in the range of cortical bone. HUVEC and hMSC laden fibrin hydrogels were printed in osteon-like patterns and cultured in vitro. A significant increase in gene expression of angiogenic markers was observed for the biomimetic scaffolds. Finally, biphasic scaffolds were implanted subcutaneously in rats. Histological analysis of explanted scaffolds showed a significant increase in the number of blood vessels per area in the 3D printed osteon-like scaffolds. The utilization of these scaffolds in constructing biomimetic osteons for bone regeneration demonstrated a promising capacity to improve neovascularization of the construct. These results indicates that proper cell orientation and scaffold design could play a critical role in neovascularization.

An in vitro intestinal platform with a self-sustaining oxygen gradient to study the human gut/microbiome interface

Abstract: An oxygen gradient formed along the length of colonic crypts supports stem-cell proliferation at the normoxic crypt base while supporting obligate anaerobe growth in the anoxic colonic lumen. Primary human colonic epithelial cells derived from human gastrointestinal stem cells were cultured within a device possessing materials of tailored oxygen permeability to produce an oxygen-depleted luminal (0.8% ± 0.1% O2) and oxygen-rich basal (11.1% ± 0.5% O2) compartment. This oxygen difference created a stable oxygen gradient across the colonic epithelial cells which remained viable and properly polarized. Facultative and obligate anaerobes Lactobacillus rhamnosus, Bifidobacterium adolescentis, and Clostridium difficile grew readily within the luminal compartment. When formed along the length of an in vitro crypt, the oxygen gradient facilitated cell compartmentalization within the crypt by enhancing confinement of the proliferative cells to the crypt base. This platform provides a simple system to create a physiological oxygen gradient across an intestinal mimic while simultaneously supporting anaerobe co-culture.

3D printed coaxial nozzles for the extrusion of hydrogel tubes toward modeling vascular endothelium

Abstract: Engineered tubular constructs made from soft biomaterials are employed in a myriad of applications in biomedical science. Potential uses of these constructs range from vascular grafts to conduits for enabling perfusion of engineered tissues and organs. The fabrication of standalone tubes or complex perfusable constructs from biofunctional materials, including hydrogels, via rapid and readily accessible routes is desirable. Here we report a methodology in which customized coaxial nozzles are 3D printed using commercially available stereolithography (SLA) 3D printers. These nozzles can be used for the fabrication of hydrogel tubes via coextrusion of two shear-thinning hydrogels: an unmodified Pluronic® F-127 (F127) hydrogel and an F127-bisurethane methacrylate (F127-BUM) hydrogel. We demonstrate that different nozzle geometries can be modeled via computer-aided design and 3D printed in order to generate tubes or coaxial filaments with different cross-sectional geometries. We were able to fabricate tubes with luminal diameters or wall thicknesses as small as ∼150 μm. Finally, we show that these tubes can be functionalized with collagen I to enable cell adhesion, and human umbilical vein endothelial cells can be cultured on the luminal surfaces of these tubes to yield tubular endothelial monolayers. Our approach could enable the rapid fabrication of biofunctional hydrogel conduits which can ultimately be utilized for engineering in vitro models of tubular biological structures.

Human platelet lysate-based nanocomposite bioink for bioprinting hierarchical fibrillar structures

Abstract: Three-dimensional (3D) bioprinting holds the promise to fabricate tissue and organ substitutes for regenerative medicine. However, the lack of bioactive inks to fabricate and support functional living constructs is one of the main limitations hindering the progress of this technology. In this study, a biofunctional human-based nanocomposite bioink (HUink) composed of platelet lysate hydrogels reinforced by cellulose nanocrystals is reported. When combined with suspended bioprinting technologies, HUink allows the biofabrication of 3D freeform constructs with high resolution and integrity, mimicking the hierarchical nano-to-macro fibrillary composition of native tissues. Remarkably, HUink supports bioprinting of stem cells with high viability immediately after extrusion and over long-term cell culture without the need for additional biochemical or animal-derived media supplementation. As opposed to typical polymer-based bioinks, the pool of growth factors, cytokines and adhesion proteins in HUink boosts cell spreading and proliferation, stimulating the fast production of cell-secreted extracellular matrix. This innovative bioprinting platform with unpaired biofunctionality allows the fabrication of complex freeform cell-laden constructs that can ultimately be applied in the development of xeno-free 3D tissue models for in vitro research or to develop tissue and organ surrogates for clinical applications.

Thermal inkjet bioprinting triggers the activation of the VEGF pathway in human microvascular endothelial cells in vitro

Abstract: One biofabrication process that has gained tremendous momentum in the field of tissue engineering and regenerative medicine is cell-printing or most commonly bioprinting. We have shown that thermal inkjet bioprinted human microvascular endothelial cells were recruited or otherwise involved in the formation of microvasculature to form graft-host anastomoses upon implantation. The present study aims to quantify and characterize the expression and activation of specific cytokines and kinases in vitro. Morphological characteristics demonstrate elongated protrusions of TIB-HMVECs at 5-6 times the size of manually pipetted cells. Moreover, annexin V-FITC and propidium iodide apoptosis assay via flow cytometry demonstrated a 75% apoptosis among printed cells as compared to among control cells. Cell viability at a 3 d incubation period was significantly higher for printed cells as compared to control. Milliplex magnetic bead panels confirmed significant overexpression of HSP70, IL-1α, VEGF-A, IL-8, and FGF-1 of printed cells compared to control. In addition, a Human phospho-kinase array displayed a significant over activation of the heat-shock proteins HSP27 and HSP60 of printed cells compared to the manually seeded cells. Collectively, it is suggested that the massive appearance of capillary blood vessels upon implantation that has been reported elsewhere may be due to the activation of the HSP-NF-κB pathway to produce VEGF. This cell activation may be used as a new strategy for vascularization of tissue engineered constructs which are in high demand in regenerative medicine applications.

Printomics: the high-throughput analysis of printing parameters applied to melt electrowriting

Abstract: Melt electrowriting (MEW) combines the fundamental principles of electrospinning, a fibre forming technology, and 3D printing. The process, however, is highly complex and the quality of the fabricated structures strongly depends on the interplay of key printing parameter settings including processing temperature, applied voltage, collection speed, and applied pressure. These parameters act in unison, comprising the principal forces on the electrified jet: pushing the viscous polymer out of the nozzle and mechanically and electrostatically dragging it for deposition towards the collector. Although previous studies interpreted the underlying mechanism of electrospinning with polymer melts in a direct writing mode, contemporary devices used in laboratory environments lack the capability to collect large data reproducibly. Yet, a validated large data set is a condition sine qua non to design an in-process control system which allows to computer control the complexity of the MEW process. For this reason, we engineered an advanced automated MEW system with monitoring capabilities to specifically generate large, reproducible data volumes which allows the interpretation of complex process parameters. Additionally, the design of an innovative real-time MEW monitoring system identifies the main effects of the system parameters on the geometry of the fibre flight path. This enables, for the first time, the establishment of a comprehensive correlation between the input parameters and the geometry of a MEW jet. The study verifies the most stable process parameters for the highly reproducible fabrication of a medical-grade poly(e-caprolactone) fibres and demonstrates how Printomics can be performed for the high throughput analysis of processing parameters for MEW.

Incorporation of cerium oxide in hollow mesoporous bioglass scaffolds for enhanced bone regeneration by activating the ERK signaling pathway.

Abstract: Hierarchically porous structures and bioactive compositions of artificial biomaterials play a positive role in bone defect healing and new bone regeneration. Herein, cerium oxide nanoparticles-modified bioglass (Ce-BG) scaffolds were firstly constructed by the incorporation of hollow mesoporous Ce-BG microspheres in CTS via a freeze-drying technology. The interconnected macropores in Ce-BG scaffolds facilitated the in-growth of bone cells/tissues from material surfaces into the interiors, while the hollow cores and mesopore shells in Ce-BG microspheres provides more active sites for bone mineralization. The cerium oxide nanoparticles in the scaffolds rapidly promoted the proliferation and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs), as confirmed by the up-regulation of osteogenesis-related markers such as OCN, ALP and COL-1. The enhanced osteoinductivity of Ce-BG scaffolds was mainly related to the activated ERK pathway, and it was blocked by adding a selective ERK1/2 inhibitor (SCH772984). In vivo rat cranial defect models revealed that Ce-BG scaffolds accelerated collagen deposition, osteoblast formation and bone regeneration as compared to BG scaffolds. The exciting results demonstrated that the synergistic effects between hierarchically porous structures and cerium oxide nanoparticles contributed to osteogenic ability, and hollow mesoporous Ce-BG scaffolds would be a novel platform for bone regeneration.

Evaluation of sterilisation methods for bio-ink components: gelatin, gelatin methacryloyl, hyaluronic acid and hyaluronic acid methacryloyl.

Abstract: Reliable and scalable sterilisation of hydrogels is critical to the clinical translation of many biofabrication approaches, such as extrusion-based 3D bioprinting of cell-laden bio-inks. However sterilisation methods can be destructive, and may have detrimental effects on the naturally-derived hydrogels that constitute much of the bio-ink palette. Determining effective sterilisation methods requires detailed analysis of the effects of sterilisation on relevant properties such as viscosity, printability and cytocompatibility. Yet there have been no studies specifically exploring the effects of sterilisation on bio-inks to date. In this work, we explored the effects of various sterilisation techniques on four of the most widely used bio-ink components: gelatin, gelatin methacryloyl, hyaluronic acid, and hyaluronic acid methacrylate. Autoclaving was the most destructive sterilisation method, producing large reductions in viscosity and in mechanical properties following crosslinking. Filter sterilisation caused some reduction in rheological properties of GelMA due to removal of higher molecular weight components, but did not affect photocrosslinking. Ethylene oxide (EtO) was the least destructive sterilisation method in terms of rheological properties for all materials, had no detrimental effect on the photocrosslinkable methacrylate/methacrylamide groups, and so was chosen for more detailed examination. In biological analyses, we found that EtO treatment successfully eradicated a bacterial challenge of E. coli, caused no decrease in viability of human mesenchyman stem cells (hMSCs), and had no effect on their rate of proliferation. Finally, we found that EtO-treated hydrogels supported encapsulated hMSCs to differentiate towards the chondrogenic lineage, and to produce new cartilage matrix. Our results bring to light various effects that sterilisation can have on bio-inks, as well as highlighting EtO sterilisation as a method which minimises degradation of properties, while still promoting biological function.

Cryogenic free-form extrusion bioprinting of decellularized small intestinal submucosa for potential applications in skin tissue engineering.

Abstract: A novel strategy of cryogenic 3D bioprinting assisted by free-from extrusion printing has been developed and applied to printing of a decellularized small intestinal submucosa (dSIS) slurry The rheological properties, including kinetic viscosity, storage modulus (G'), and loss modulus (G″), were appropriate for free-from extrusion printing of dSIS slurry Three different groups of scaffolds, including P500, P600, and P700, with filament distances of 500, 600, and 700 μm, respectively were fabricated at a 5 mm s-1 working velocity of the platform (V xy) and 25 kPa air pressure of the dispensing system (P) at -20 °C The fabricated scaffolds were crosslinked via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) which resulted in a polyporous microstructure The variations in the filament diameter and pore size were evaluated in the initial frozen state after printing, the lyophilized state, and after immersion in a PBS solution The Young's modulus of the P500, P600, and P700 scaffolds was measured in wet and dry states for EDC-crosslinked scaffolds The cell experiment results showed improved cell adhesion, viability, and proliferation both on the surface and within the scaffold, indicating the biocompatibility and suitability of the scaffold for 3D cell models Further, gene and protein expression of normal skin fibroblasts on dSIS scaffolds demonstrated their ability to promote the production of some extracellular matrix proteins (ie collagen I, collagen III, and fibronectin) in vitro Overall, this study presents a new potential strategy, by combining cryogenic 3D bioprinting with decellularized extracellular matrix materials, to manufacture ideal scaffolds for skin tissue engineering applications