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  • 3D printing of chemically crosslinked gelatin hydrogels for adipose tissue engineering.
    Biofabrication (IF 7.236) Pub Date : 2019-11-13
    Nicola Contessi Negrini,Nehar Celikkin,Paolo Tarsini,Silvia Fare,Wojciech Święszkowski

    Despite the outstanding potential and success achieved in 3D printed hydrogel scaffolds, their application in regeneration of damaged or missing adipose tissue (AT) has yet been poorly investigated. Thanks to the desired macroscopic shape, microarchitecture, extracellular matrix mimicking structure, degradability and soft tissue biomimetic mechanical properties, 3D printed hydrogel scaffolds possess a great potential of simultaneously targeting aesthetic, structural and functional AT restoration. Here, we propose a simple and cost-effective 3D printing strategy of a gelatin-based ink to fabricate scaffolds suitable for AT engineering. The ink, successfully printed here for the first time, was prepared by mixing gelatin and methylenebisacrylamide (i.e., crosslinker) to initiate the crosslinking reaction. The solution was then loaded in the cartridge (temperature T = 35 °C) of a pneumatic extrusion-based 3D printer and printed on a cooled surface (T = 4 °C) in the appropriate ink printability time window verified by rheological tests. Subsequently, the printed gelatin hydrogels were crosslinked at different temperatures to optimize their stability and fix the printed structure. The gelatin scaffolds crosslinked at 20 °C remained stable for 21 days at physiological temperature, with compressive mechanical properties suitable to mimic those of AT (i.e., elastic modulus = 20 kPa). The 3D printed scaffolds showed no indirect cytotoxic effects for 3T3-L1 preadipocyte cells line. Moreover, the printed scaffolds successfully promoted primary human preadipocytes adhesion and proliferation, as demonstrated by LIVE/DEAD staining and Alamar Blue assay. Besides, the differentiation of primary human preadipocytes isolated from three different donors towards adipogenic phenotype was demonstrated by an increase in PPARγ gene expression detected by real time PCR and accumulated lipid droplets stained by Oil Red O, thus proving the potential of the 3D printed gelatin hydrogels as scaffolds for AT engineering.

    更新日期:2020-01-16
  • Bioprinted trachea constructs with patient-matched design, mechanical and biological properties
    Biofabrication (IF 7.236) Pub Date : 2019-12-31
    Dongxu Ke, Hualin Yi, Savannah Est-Witte, Sunil George, Carlos Kengla, Sang Jin Lee, Anthony Atala and Sean V Murphy

    Tracheal stenosis is a rare but life-threatening disease. Primary clinical procedures for treating this disease are limited if the region requiring repair is long or complex. This study is the first of its kind to fabricate bioprinted tracheal constructs with separate cartilage and smooth muscle regions using polycaprolactone (PCL) and human mesenchymal stem cell (hMSC)-laden hydrogels. Our final bioprinted trachea showed comparable elastic modulus and yield stress compared to native tracheal tissue. In addition, both cartilage and smooth muscle formation were observed in the desired regions of our bioprinted trachea through immunohistochemistry and western blot after two weeks of in vitro culture. This study demonstrates a novel approach to manufacture tissue engineered trachea with mechanical and biological properties similar to native trachea, which represents a step closer to overcoming the clinical challenges of treating tracheal stenosis.

    更新日期:2019-12-31
  • Engineering bioprintable alginate/gelatin composite hydrogels with tunable mechanical and cell adhesive properties to modulate tumor spheroid growth kinetics
    Biofabrication (IF 7.236) Pub Date : 2019-12-31
    Tao Jiang, Jose G Munguia-Lopez, Kevin Gu, Maeva M Bavoux, Salvador Flores-Torres, Jacqueline Kort-Mascort, Joel Grant, Sanahan Vijayakumar, Antonio De Leon-Rodriguez, Allen J Ehrlicher and Joseph M Kinsella

    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 concentrati...

    更新日期:2019-12-31
  • Tuning the mechanics of 3D-printed scaffolds by crystal lattice-like structural design for breast tissue engineering
    Biofabrication (IF 7.236) Pub Date : 2019-12-31
    Muran Zhou, Jinfei Hou, Guo Zhang, Chao Luo, Yuyang Zeng, Shan Mou, Peng Xiao, Aimei Zhong, Quan Yuan, Jie Yang, Zhenxing Wang and Jiaming Sun

    Breast tissue engineering is a promising alternative to standard treatments for breast defects. Although there is a consensus that the mechanical property of the scaffold should best match the reconstructed tissue, the simulation of the soft and elastic tactility of native breast tissues using conventional materials and architecture design requires further study. Previous research has shown that the crystal microstructure-like design can drastically alter the mechanical properties of the constructed scaffolds. In this study, we designed and additive manufactured four kinds of breast scaffolds using polyurethane and termed their architectures as N5S4, N9S8, N7S6 and N4S6. The basic unit cell of each scaffold was similar to a lattice structure from the isometric crystal system. The scaffolds possessed identical porosity but different mechanical properties in which the compressive modulus of the softest scaffolds (N5S4) were similar to that of native breast tissue. When applied in ...

    更新日期:2019-12-31
  • Enhanced mechanical and electrical properties of heteroscaled hydrogels infused with aqueous-dispersible hybrid nanofibers
    Biofabrication (IF 7.236) Pub Date : 2019-12-19
    Suntae Kim and Chaenyung Cha

    Despite the widespread use as platforms for various biomedical applications, engineering hydrogels to impart multifunctionality and control physical properties, while closely mimicking the native cellular microenvironment, is still a significant challenge. Herein, nanofibers consisting of hydrophilic and photocrosslinkable biopolymer and conductive polymer (i.e. PEDOT:PSS) are first fabricated via electrospinning, cut into micrometer-lengths, and chemically crosslinked to develop dispersible hybrid nanofiber (dhNF) as heteroscale reinforcing elements for developing nanocomposite hydrogels. The dhNF can be readily dispersed in aqueous precursor solutions without dissolution and incorporated into hydrogels. The resulting ‘heteroscale’ dhNF-infused hydrogels, consisting of molecular and nanofibrous polymeric network, more closely resembles natural extracellular matrix, and show significant improvement on both mechanical strength and electrical conductivity, by dhNF concentration as...

    更新日期:2019-12-19
  • Scalable and cost-effective generation of osteogenic micro-tissues through the incorporation of inorganic microparticles within mesenchymal stem cell spheroids
    Biofabrication (IF 7.236) Pub Date : 2019-12-19
    Ibrahim Zarkesh, Majid Halvaei, Mohammad Hossein Ghanian, Fatemeh Bagheri, Forough Azam Sayahpour, Mahmoud Azami, Javad Mohammadi, Hossein Baharvand and Mohamadreza Baghaban Eslaminejad

    Mesenchymal stem cells (MSCs) are considered primary candidates for treating complex bone defects in cell-based therapy and tissue engineering. Compared with monolayer cultures, spheroid cultures of MSCs (mesenspheres) are favorable due to their increased potential for differentiation, extracellular matrix (ECM) synthesis, paracrine activity, and in vivo engraftment. Here, we present a strategy for the incorporation of microparticles for the fabrication of osteogenic micro-tissues from mesenspheres in a cost-effective and scalable manner. A facile method was developed to synthesize mineral microparticles with cell-sized spherical shape, biphasic calcium phosphate composition (hydroxyapatite and β -tricalcium phosphate), and a microporous structure. Calcium phosphate microparticles (CMPs) were incorporated within the mesenspheres through mixing with the single cells during cell aggregation. Interestingly, the osteogenic genes were upregulated significantly (collagen ...

    更新日期:2019-12-19
  • 4D biofabrication of skeletal muscle microtissues
    Biofabrication (IF 7.236) Pub Date : 2019-12-11
    Indra Apsite, Juan Manuel Uribe, Andrés Fernando Posada, Sabine Rosenfeldt, Sahar Salehi and Leonid Ionov

    Skeletal muscle is one of the most abundant tissues in the body. Although it has a relatively good regeneration capacity, it cannot heal in the case of disease or severe damage. Many current tissue engineering strategies fall short due to the complex structure of skeletal muscle. Biofabrication techniques have emerged as a popular set of methods for increasing the complexity of tissue-like constructs. In this paper, 4D biofabrication technique is introduced for fabrication of the skeletal muscle microtissues. To this end, a bilayer scaffold consisting of a layer of anisotropic methacrylated alginate fibers (AA-MA) and aligned polycaprolactone (PCL) fibers were fabricated using electrospinning and later induced to self-fold to encapsulate myoblasts. Bilayer mats undergo shape-transformation in an aqueous buffer, a process that depends on their overall thickness, the thickness of each layer and the geometry of the mat. Proper selection of these parameters allowed fabrication of sc...

    更新日期:2019-12-13
  • The development of a high throughput drug-responsive model of white adipose tissue comprising adipogenic 3T3-L1 cells in a 3D matrix
    Biofabrication (IF 7.236) Pub Date : 2019-12-11
    Alexander D Graham, Rajesh Pandey, Viktoriya S Tsancheva, Alessia Candeo, Stanley W Botchway, Alasdair J Allan, Lydia Teboul, Kamel Madi, Tahkur S Babra, Louisa A K Zolkiewski, Xuan Xue, Liz Bentley, Joan Gannon, Sam N Olof and Roger D Cox

    Adipose models have been applied to mechanistic studies of metabolic diseases (such as diabetes) and the subsequent discovery of new therapeutics. However, typical models are either insufficiently complex (2D cell cultures) or expensive and labor intensive (mice/ in vivo ). To bridge the gap between these models and in order to better inform pre-clinical studies we have developed a drug-responsive 3D model of white adipose tissue (WAT). Here, spheroids (680 ± 60 μ m) comprising adipogenic 3T3-L1 cells encapsulated in 3D matrix were fabricated manually on a 96 well scale. Spheroids were highly characterised for lipid morphology, selected metabolite and adipokine secretion, and gene expression; displaying significant upregulation of certain adipogenic-specific genes compared with a 2D model. Furthermore, induction of lipolysis and promotion of lipogenesis in spheroids could be triggered by exposure to 8-br-cAMP and oleic-acid respectively. Metabolic and high content ima...

    更新日期:2019-12-13
  • Direct process feedback in extrusion-based 3D bioprinting
    Biofabrication (IF 7.236) Pub Date : 2019-12-11
    Ashley A Armstrong, Julian Norato, Andrew G Alleyne and Amy J Wagoner Johnson

    A major limitation in extrusion-based bioprinting is the lack of direct process control, which limits the accuracy and design complexity of printed constructs. The lack of direct process control results in a number of defects that can influence the functional and mechanical outcomes of the fabricated structures. The machine axes motion cannot be reliably used to predict material placement, and precise fabrication requires additional sensing of the material extrusion. We present an iteration-to-iteration process monitoring system that enables direct process control in the material deposition reference frame. To fabricate parts with low dimensional errors, we integrate a non-contact laser displacement scanner into the printing platform. After fabrication of the initial print using the as-designed reference trajectory, the laser scanner moves across the part to measure the material placement. A custom image processing algorithm compares the laser scanner data to the as-designed ref...

    更新日期:2019-12-13
  • Integration of an ultra-strong poly(lactic-co-glycolic acid) (PLGA) knitted mesh into a thermally induced phase separation (TIPS) PLGA porous structure to yield a thin biphasic scaffold suitable for dermal tissue engineering
    Biofabrication (IF 7.236) Pub Date : 2019-12-04
    Eamonn McKenna, Travis J Klein, Michael R Doran and Kathryn Futrega

    We aimed to capture the outstanding mechanical properties of meshes, manufactured using textile technologies, in thin biodegradable biphasic tissue-engineered scaffolds through encapsulation of meshes into porous structures formed from the same polymer. Our novel manufacturing process used thermally induced phase separation (TIPS), with ethylene carbonate (EC) as the solvent, to encapsulate a poly(lactic-co-glycolic acid) (PLGA) mesh into a porous PLGA network. Biphasic scaffolds (1 cm × 4 cm × 300 μ m) were manufactured by immersing strips of PLGA mesh in 40 °C solutions containing 5% PLGA in EC, supercooling at 4 °C for 4 min, triggering TIPS by manually agitating the supercooled solution, and lastly eluting EC into 4 °C Milli-Q water. EC processing was rapid and did not compromise mesh tensile properties. Biphasic scaffolds exhibited a tensile strength of 40.7 ± 2.2 MPa, porosity of 94%, pore size of 16.85 ± 3.78 μ m, supported HaCaT cell proliferation, and degrad...

    更新日期:2019-12-04
  • Glial cells influence cardiac permittivity as evidenced through in vitro and in silico models
    Biofabrication (IF 7.236) Pub Date : 2019-12-02
    Jonathan R Soucy, Jody Askaryan, David Diaz, Abigail N Koppes, Nasim Annabi and Ryan A Koppes

    Excitation–contraction (EC) coupling in the heart has, until recently, been solely accredited to cardiomyocytes. The inherent complexities of the heart make it difficult to examine non-muscle contributions to contraction in vivo, and conventional in vitro models fail to capture multiple features and cellular heterogeneity of the myocardium. Here, we report on the development of a 3D cardiac μ Tissue to investigate changes in the cellular composition of native myocardium in vitro . Cells are encapsulated within micropatterned gelatin-based hydrogels formed via visible light photocrosslinking. This system enables spatial control of the microarchitecture, perturbation of the cellular composition, and functional measures of EC coupling via video microscopy and a custom algorithm to quantify beat frequency and degree of coordination. To demonstrate the robustness of these tools and evaluate the impact of altered cell population densities on cardiac μ T...

    更新日期:2019-12-02
  • Fast three-dimensional micropatterning of PC12 cells in rapidly crosslinked hydrogel scaffolds using ultrasonic standing waves
    Biofabrication (IF 7.236) Pub Date : 2019-12-02
    Kai W Cheng, Layla Alhasan, Amgad R Rezk, Aswan Al-Abboodi, Pauline M Doran, Leslie Y Yeo and Peggy P Y Chan

    The ability to spatially organise the microenvironment of tissue scaffolds unlocks the potential of many scaffold-based tissue engineering applications. An example application is to aid the regeneration process of peripheral nerve injuries. Herein, we present a promising approach for three-dimensional (3D) micropatterning of nerve cells in tissue scaffolds for peripheral nerve repair. In particular, we demonstrate the 3D micropatterning of PC12 cells in a gelatin-hydroxyphenylpropionic acid (Gtn-HPA) hydrogel using ultrasound standing waves (USWs). PC12 cells were first aligned in 3D along nodal planes by the USWs in Gtn-HPA hydrogel precursor solution. The precursor was then crosslinked using horseradish peroxidase (HRP) and diluted hydrogen peroxide (H 2 O 2 ), thus immobilising the aligned cells within 90–120 s. This micropatterning process is cost effective and can be replicated easily without the need for complex and expensive specialised equipment. USW-al...

    更新日期:2019-12-02
  • Human platelet lysate-based nanocomposite bioink for bioprinting hierarchical fibrillar structures
    Biofabrication (IF 7.236) Pub Date : 2019-11-27
    Bárbara B Mendes, Manuel Gómez-Florit, Alex G Hamilton, Michael S Detamore, Rui M A Domingues, Rui L Reis and Manuela E Gomes

    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 p...

    更新日期:2019-11-29
  • 3D bioprinting of liver spheroids derived from human induced pluripotent stem cells sustain liver function and viability in vitro
    Biofabrication (IF 7.236) Pub Date : 2019-11-27
    Ernesto Goulart, Luiz Carlos de Caires-Junior, Kayque Alves Telles-Silva, Bruno Henrique Silva Araujo, Silvana Aparecida Rocco, Mauricio Sforca, Irene Layane de Sousa, Gerson S Kobayashi, Camila Manso Musso, Amanda Faria Assoni, Danyllo Oliveira, Elia Caldini, Silvano Raia, Peter I Lelkes and Mayana Zatz

    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/amin...

    更新日期:2019-11-29
  • Endothelialized microrods for minimally invasive in situ neovascularization
    Biofabrication (IF 7.236) Pub Date : 2019-11-27
    Ying Wang, Xuan Hu, Ranjith Kumar Kankala, Da-Yun Yang, Kai Zhu, Shi-Bin Wang, Yu Shrike Zhang and Ai-Zheng Chen

    Despite the significant advancements in fabricating various scaffolding systems over the past decades, generation of functional tissues towards vascularization remains challenging for the currently available biofabrication approaches. On the other hand, the applicability of traditional surgical transplantation of vascularized tissue constructs is sometimes limited due to the sophisticated surgical procedures, which are invasive, leading to increased risks of scar formation and infection. Considering these facts, we present an innovative platform, the angiogenic microrods composed of sodium alginate/gelatin harboring proliferating endothelial cells using a specially designed double T-junction microfluidic device with an expansion chamber, for achieving minimally invasive neovascularization in situ . Such vessel-like microarchitectures could be derived through controlled penetration of the crosslinker genepin for the gelatin phase, ensuing differential degrees in crosslinkin...

    更新日期:2019-11-29
  • In situ tissue engineering of the tendon-to-bone interface by endogenous stem/progenitor cells
    Biofabrication (IF 7.236) Pub Date : 2019-11-18
    Solaiman Tarafder, John A Brito, Sumeet Minhas, Linda Effiong, Stavros Thomopoulos and Chang H Lee

    The long-term success of surgical repair of rotator cuff tears is largely dependent on restoration of a functional tendon-to-bone interface. We implemented micro-precise spatiotemporal delivery of growth factors in three-dimensional printed scaffolds for integrative regeneration of a fibrocartilaginous tendon-to-bone interface. Sustained and spatially controlled release of tenogenic, chondrogenic and osteogenic growth factors was achieved using microsphere-based delivery carriers embedded in thin membrane-like scaffolds. In vitro , the scaffolds embedded with spatiotemporal delivery of growth factors successfully guided regional differentiation of mesenchymal progenitor cells, forming multiphase tissues with tendon-like, cartilage-like and bone-like regions. In vivo , when implanted at the interface between the supraspinatus tendon and the humeral head in a rat rotator cuff repair model, these scaffolds promoted recruitment of endogenous tendon progenitor cells follo...

    更新日期:2019-11-18
  • Layer-specific cell differentiation in bi-layered vascular grafts under flow perfusion
    Biofabrication (IF 7.236) Pub Date : 2019-11-18
    Iris Pennings, Eline E van Haaften, Tomasz Jungst, Jurgen A Bulsink, Antoine J W P Rosenberg, Jürgen Groll, Carlijn V C Bouten, Nicholas A Kurniawan, Anthal I P M Smits and Debby Gawlitta

    Bioengineered grafts have the potential to overcome the limitations of autologous and non-resorbable synthetic vessels as vascular substitutes. However, one of the challenges in creating these living grafts is to induce and maintain multiple cell phenotypes with a biomimetic organization. Our biomimetic grafts with heterotypic design hold promises for functional neovessel regeneration by guiding the layered cellular and tissue organization into a native-like structure. In this study, a perfusable two-compartment bioreactor chamber was designed for the further maturation of these vascular grafts, with a compartmentalized exposure of the graft’s luminal and outer layer to cell-specific media. We used the system for a co-culture of endothelial colony forming cells and multipotent mesenchymal stromal cells (MSCs) in the vascular grafts, produced by combining electrospinning and melt electrowriting. It was demonstrated that the targeted cell phenotypes (i.e. endothelial cells (ECs) a...

    更新日期:2019-11-18
  • An in vitro intestinal platform with a self-sustaining oxygen gradient to study the human gut/microbiome interface
    Biofabrication (IF 7.236) Pub Date : 2019-11-06
    Raehyun Kim, Peter J Attayek, Yuli Wang, Kathleen L Furtado, Rita Tamayo, Christopher E Sims and Nancy L Allbritton

    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% O 2 ) and oxygen-rich basal (11.1% ± 0.5% O 2 ) 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 t...

    更新日期:2019-11-06
  • Collagen/bioceramic-based composite bioink to fabricate a porous 3D hASCs-laden structure for bone tissue regeneration
    Biofabrication (IF 7.236) Pub Date : 2019-11-06
    WonJin Kim and GeunHyung Kim

    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 proliferatio...

    更新日期:2019-11-06
  • Three-dimensional cartilage tissue regeneration system harnessing goblet-shaped microwells containing biocompatible hydrogel.
    Biofabrication (IF 7.236) Pub Date : 2019-11-30
    Nopphadol Udomluck,Sung-Hwan Kim,Hyunjoo Cho,Joong Yull Park,Hansoo Park

    Differentiation of stem cells into chondrocytes has been studied for the engineering of cartilage tissue. However, stem cells cultured two-dimensionally have limited ability to differentiate into chondrocytes, which led to the development of three-dimensional culture systems. A recently developed microtechnological method uses microwells as a tool to form uniformly sized spheroids. In this study, we fabricated an array (10 × 10) of goblet-shaped microwells based on polydimethylsiloxane for spheroid culture. A central processing unit (CPU) was used to form holes, and metallic beads were used to form hemispherical microwell geometry. The holes were filled with Pluronic F-127 to prevent cells from sinking through the holes and allowing the cells to form spheroids. Viability and chondrogenic differentiation of human adipose-derived stem cells were assessed. The fabrication method using a micro-pin mold and metallic beads is easy and cost-effective. Our three-dimensional spheroid culture system optimizes the efficient differentiation of cells and has various applications, such as drug delivery, cell therapy, and tissue engineering.

    更新日期:2019-11-01
  • Decellularized extracellular matrix-based bio-ink with enhanced 3D printability and mechanical properties.
    Biofabrication (IF 7.236) Pub Date : 2019-11-30
    Min Kyeong Kim,Wonwoo Jeong,Sang Min Lee,Jeong Beom Kim,Songwan Jin,Hyun-Wook Kang

    Recently, decellularized extracellular matrix-based bio-ink (dECM bio-ink) derived from animal organs is attracting attention because of its excellent biocompatibility. However, its poor 3D printability and weak mechanical properties remain a challenge. Here, we developed a new dECM bio-ink with enhanced 3D printability and mechanical properties. dECM micro-particles of about 13.4 μm in size were prepared by decellularizing a porcine liver followed by freeze-milling. The new bio-ink, named as dECM powder-based bio-ink (dECM pBio-ink), was prepared by loading the dECM micro-particles into a gelatin mixture. The usefulness of the dECM pBio-ink was evaluated by assessing its mechanical properties, printability, and cytocompatibility. The results showed that its mechanical properties and 3D printability were greatly improved. Its elastic modulus increased by up to 9.17 times that of the conventional dECM bio-ink. Micro-patterns with living cells were successfully achieved with 93 % cell viability. Above all, the new bio-ink showed superior performance in stacking of layers for 3D printing, whereas the conventional bio-ink could not maintain its shape. Finally, we demonstrated that the dECM pBio-ink possessed comparable cytocompatibility with the conventional dECM bio-ink through in-vitro tests with endothelial cells and primary mouse hepatocytes.

    更新日期:2019-11-01
  • Customizable, engineered substrates for rapid screening of cellular cues.
    Biofabrication (IF 7.236) Pub Date : 2019-11-30
    Eline Huethorst,Marie F A Cutiongco,Fraser A Campbell,Anwer Saeed,Rachel Love,Paul M Reynolds,Matthew J Dalby,Nikolaj Gadegaard

    Biophysical cues robustly direct cell responses and are thus important tools for in vitro and translational biomedical applications. High throughput platforms exploring substrates with varying physical properties are therefore valuable. However, currently existing platforms are limited in throughput, the biomaterials used, the capability to segregate between different cues and the assessment of dynamic responses. Here we present a multiwell array (3x8) made of a substrate engineered to present topography or rigidity cues welded to a bottomless plate with a 96-well format. Both the patterns on the engineered substrate and the well plate format can be easily customized, permitting systematic and efficient screening of biophysical cues. To demonstrate the broad range of possible biophysical cues examinable, we designed and tested three multiwell arrays to influence cardiomyocyte, chondrocyte and osteoblast function. Using the multiwell array, we were able to measure different cell functionalities using analytical modalities such as live microscopy, qPCR and immunofluorescence. We observed that grooves (5 µm in size) induced less variation in contractile function of cardiomyocytes. Compared to unpatterned plastic, nanopillars with 127 nm height, 100 nm diameter and 300 nm pitch enhanced matrix deposition, chondrogenic gene expression and chondrogenic maintenance. High aspect ratio pillars with an elastic shear modulus of 16 kPa mimicking the matrix found in early stages of bone development improved osteogenic gene expression compared to stiff plastic. We envisage that our bespoke multiwell array will accelerate the discovery of relevant biophysical cues through improved throughput and variety.

    更新日期:2019-11-01
  • Aerosol jet printing of biological inks by ultrasonic delivery.
    Biofabrication (IF 7.236) Pub Date : null
    Nicholas X Williams,Nathan Watson,Daniel Y Joh,Ashutosh Chilkoti,Aaron D Franklin

    Printing is a promising method to reduce the cost of fabricating biomedical devices. While there have been significant advancements in direct-write printing techniques, non-contact printing of biological reagents has been almost exclusively limited to inkjet printing. Motivated by this lacuna, this work investigated aerosol jet printing of biological reagents onto a nonfouling polymer brush to fabricate in vitro diagnostic (IVD) assays. The ultrasonication ink delivery process, which had previously been reported to damage DNA molecules, caused no degradation of printed proteins, allowing printing of a streptavidin-biotin binding assay with sub-nanogram mL-1 analytical sensitivity. Furthermore, a carcinoembryogenic antigen (CEA) IVD was printed and found to have sensitivities in the clinically relevant range (limit of detection of approximately 0.5 ng mL-1 and a dynamic range of approximately 3 orders of magnitude). Finally, the multi-material printing capabilities of the aerosol jet printer were demonstrated by printing silver nanowires and streptavidin as interconnected patterns in the same print job without removal of the substrate from the printer, which will facilitate the fabrication of mixed-material devices. As cost, versatility, and ink usage become more prominent factors in the development of IVDs, this work has shown that aerosol jet printing should become a more widely considered technique for fabrication.

    更新日期:2019-11-01
  • 3D printing of metal-organic framework nanosheets-structured scaffolds with tumor therapy and bone construction.
    Biofabrication (IF 7.236) Pub Date : 2019-11-23
    Wentao Dang,Bing Ma,Bo Li,Zhiguang Huan,Nan Ma,Haibo Zhu,Jiang Chang,Yin Xiao,Chengtie Wu

    After surgical resection for bone tumor, the uncleared bone tumor cells can multiply and cause recurrence of bone tumor. It is worthwhile to design a scaffold that kills the remaining bone tumor and repairs bone defects that were given rise to by surgical resection. Additionally, it is extremely important to consider the function of angiogenesis at the process of bone regeneration because the newly-formed blood vessel can offer the nutrients for bone regeneration. In this work, a novel scaffold that is metal-organic framework Cu-TCPP nanosheets interface-structured β-tricalcium phosphate (TCP) scaffold (Cu-TCPP-TCP) was successfully prepared through integrating 3D printing technique with in situ growth method in a solvothermal system. Owing to the excellent photothermal effect of Cu-TCPP nanosheets, Cu-TCPP-TCP scaffolds that were illuminated by near infrared (NIR) light demonstrated photothermal performance, which was well regulated through varying the contents of Cu-TCPP nanosheets, ambient humidity and power density of NIR light. When cultured with osteosarcoma cells, Cu-TCPP-TCP scaffolds significantly killed osteosarcoma cells through the released heat energy. Similarly, Cu-TCPP-TCP scaffolds ablated the subcutaneous bone tumor tissues at the backs of naked mice and suppressed their growth because of transformed heat energy from NIR light. The in vitro studies found that Cu-TCPP-TCP scaffolds well supported the attachments of both human bone marrow stromal cells (HBMSCs) and human umbilical vein endothelial cells (HUVECs), and significantly stimulated expressions of osteogenesis differentiation-related genes in HBMSCs and angiogenesis differentiation-related genes in HUVECs. After implanting Cu-TCPP-TCP scaffolds into the bone defects of rabbits, they effectively promoted bone regeneration. Thus, the integration of the bone-forming bioactivity of TCP scaffolds with the photothermal property of Cu-TCPP nanosheets and angiogenesis activity of Cu ions awards Cu-TCPP-TCP scaffolds with multifunctions, representing a new horizon to develop biomaterials for simultaneously curing bone tumor and repairing bone defects.

    更新日期:2019-11-01
  • Bioprinted osteon-like scaffolds enhance in vivo neovascularization.
    Biofabrication (IF 7.236) Pub Date : 2019-02-16
    Charlotte Piard,Hannah Baker,Timur Kamalitdinov,John Fisher

    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.

    更新日期:2019-11-01
  • Disturbed flow disrupts the blood-brain barrier in a 3D bifurcation model.
    Biofabrication (IF 7.236) Pub Date : 2019-11-19
    Nesrine Bouhrira,Brandon Jude DeOre,Daniel Warren Sazer,Zakary Chiaradia,Jordan S Miller,Peter A Galie

    The effect of disturbed flow profiles on the endothelium have been studied extensively in systemic vasculature, but less is known about the response of the blood-brain barrier (BBB) to these flow regimes. Here we investigate the effect of disturbed flow on the integrity of the BBB using a three-dimensional, perfusable bifurcation model consisting of a co-culture of endothelial cells with mural and glial cells. Experimental flow patterns predicted by computational fluid dynamics mimic in vivo flow regimes, specifically the presence of a recirculation zone immediately downstream of the bifurcation. Dextran permeability assays and immunostaining with markers for tight junctions show that barrier disruption is significantly greater in areas of disturbed flow compared to fully developed regions downstream of the bifurcation. Probing crosstalk between cell types suggests that disturbed flow causes barrier breakdown independent of endothelial-mural and endothelial-glial interaction. Overall, disturbed flow-induced disruption of the blood-brain barrier suggests that flow-mediated mechanisms may contribute to vascular pathologies in the central nervous system.

    更新日期:2019-11-01
  • Micro/nanofabrication of brittle hydrogels using 3D printed soft ultrafine fiber molds for damage-free demolding.
    Biofabrication (IF 7.236) Pub Date : 2019-11-15
    Shang Lv,Jing Nie,Qing Gao,Chaoqie Xie,Lu-Yu Zhou,Jingjiang Qiu,Jianzhong Fu,Xin Zhao,Yong He

    Hydrogels are very popular in biomedical areas for their extraordinary biocompatibility. However, most bio-hydrogels are too brittle to perform micro/nanofabrication. An effective method is cast molding; yet during this process, many defects occur as the excessive demolding stress damages the brittle hydrogels. Here, we propose a brand-new damage-free demolding method and a soft ultrafine fiber mold (SUFM) to replace the traditional mold. Both mechanical and finite element analysis (FEA) reveal that SUFMs have obvious advantages especially when the contact area between hydrogel and mold gets larger. By means of a high-resolution 3D printing called electrohydrodynamic (EHD) printing, SUFMs with various topological structures can be achieved with the fiber diameter ranging from 500 nm to 100 μm at a low cost. Microfluidics and cell patterns are implemented as the demonstration for potential applications. Owing to the tiny scale of microstructures and the hydrophilicity of hydrogels, significant capillary effect occurs which can be utilized to deliver liquid and cells autonomously and to seed cells into those ultrafine channels evenly. The results open up a new avenue for a wider use of hydrogels in biomedical devices, tissue engineering, microfluidics and wearable electronics; the proposed fabrication method also has the potential to become a universal technique for micro/nanofabrication of brittle materials.

    更新日期:2019-11-01
  • Optimization of collagen type I-hyaluronan hybrid bioink for 3D bioprinted liver microenvironments.
    Biofabrication (IF 7.236) Pub Date : 2018-10-03
    Andrea Mazzocchi,Mahesh Devarasetty,Richard Huntwork,Shay Soker,Aleksander Skardal

    Current 3D printing of tissue is restricted by the use of biomaterials that do not recapitulate the native properties of the extracellular matrix (ECM). These restrictions have thus far prevented optimization of composition and structure of the in vivo tissue microenvironment. The artificial nature of currently used biomaterials affects cellular phenotype and function of the bioprinted tissues, and results in inaccurate modeling of disease and drug metabolism significantly. Collagen type I is the major structural component in the ECM, and is widely used as a 3D hydrogel, but is less applicable for 3D bioprinting due to low viscosity and slow polymerization. We have hypothesized that a combination of hyaluronic acid with collagen I yields a bioink with the properties required for extrusion bioprinting, while supporting native cell-matrix interactions and preservation of the native microenvironment properties. To test this hypothesis, we tested the viscoelastic properties of three bioink formulations -2:1, 3:1, and 4:1 collagen type I to hyaluronic acid, and examined cellular behavior in order to determine an optimal formulation that allows for bioprinting while supporting biological activity. We then employed this formulation to bioprint 3D liver tissue constructs containing primary human hepatocytes and liver stellate cells and tested the effects of acetaminophen, a common liver toxicant. Our results have shown that the combination of methacrylated collagen type I and thiolated hyaluronic acid yield a simple, printable bioink that allows for modulation that was directly related to stromal cell elongation. Further, the bioink adequately allowed for implementation as a support hydrogel for hepatocytes which were able to remain viable over two weeks and responded to drug treatment appropriately.

    更新日期:2019-11-01
  • Mesenchymal stem cells support growth and organization of host-liver colorectal-tumor organoids and possibly resistance to chemotherapy.
    Biofabrication (IF 7.236) Pub Date : 2017-06-08
    Mahesh Devarasetty,Edina Wang,Shay Soker,Aleksander Skardal

    Despite having yielded extensive breakthroughs in cancer research, traditional 2D cell cultures have limitations in studying cancer progression and metastasis and screening therapeutic candidates. 3D systems can allow cells to grow, migrate, and interact with each other and the surrounding matrix, resulting in more realistic constructs. Furthermore, interactions between host tissue and developing tumors influence the susceptibility of tumors to drug treatments. Host-liver colorectal-tumor spheroids composed of primary human hepatocytes, mesenchymal stem cells (MSC) and colon carcinoma HCT116 cells were created in simulated microgravity rotating wall vessel (RWV) bioreactors. The cells were seeded on hyaluronic acid-based microcarriers, loaded with liver-specific growth factors and ECM components. Only in the presence of MSC, large tumor foci rapidly formed inside the spheroids and increased in size steadily over time, while not greatly impacting albumin secretion from hepatocytes. The presence of MSC appeared to drive self-organization and formation of a stroma-like tissue surrounding the tumor foci and hepatocytes. Exposure to a commonly used chemotherapeutic 5-FU showed a dose-dependent cytotoxicity. However, if tumor organoids were allowed to mature in the RWV, they were less sensitive to the drug treatment. These data demonstrate the potential utility of liver tumor organoids for cancer progression and drug response modeling.

    更新日期:2019-11-01
  • Ultrathin transparent membranes for cellular barrier and co-culture models.
    Biofabrication (IF 7.236) Pub Date : 2017-02-01
    Robert N Carter,Stephanie M Casillo,Andrea R Mazzocchi,Jon-Paul S DesOrmeaux,James A Roussie,Thomas R Gaborski

    Typical in vitro barrier and co-culture models rely upon thick semi-permeable polymeric membranes that physically separate two compartments. Polymeric track-etched membranes, while permeable to small molecules, are far from physiological with respect to physical interactions with co-cultured cells and are not compatible with high-resolution imaging due to light scattering and autofluorescence. Here we report on an optically transparent ultrathin membrane with porosity exceeding 20%. We optimize deposition and annealing conditions to create a tensile and robust porous silicon dioxide membrane that is comparable in thickness to the vascular basement membrane (100-300 nm). We demonstrate that human umbilical vein endothelial cells (HUVECs) spread and proliferate on these membranes similarly to control substrates. Additionally, HUVECs are able to transfer cytoplasmic cargo to adipose-derived stem cells when they are co-cultured on opposite sides of the membrane, demonstrating its thickness supports physiologically relevant cellular interactions. Lastly, we confirm that these porous glass membranes are compatible with lift-off processes yielding membrane sheets with an active area of many square centimeters. We believe that these membranes will enable new in vitro barrier and co-culture models while offering dramatically improved visualization compared to conventional alternatives.

    更新日期:2019-11-01
  • Engineering of microscale vascularized fat that responds to perfusion with lipoactive hormones.
    Biofabrication (IF 7.236) Pub Date : 2018-10-05
    Xuanyue Li,Jingyi Xia,Calin T Nicolescu,Miles W Massidda,Tyler J Ryan,Joe Tien

    Current methods to treat large soft-tissue defects mainly rely on autologous transfer of adipocutaneous flaps, a method that is often limited by donor site availability. Engineered vascularized adipose tissues can potentially be a viable and readily accessible substitute to autologous flaps. In this study, we engineered a small-scale adipose tissue with pre-patterned vasculature that enables immediate perfusion. Vessels formed after one day of perfusion and displayed barrier function after three days of perfusion. Under constant perfusion, adipose tissues remained viable and responded to lipoactive hormones insulin and epinephrine with lipid accumulation and loss, respectively. Adipocyte growth correlated inversely with distance away from the feeding vessel, as predicted by a Krogh-type model.

    更新日期:2019-11-01
  • Fabrication of a three-dimensional tissue model microarray using laser foaming of a gas-impregnated biodegradable polymer.
    Biofabrication (IF 7.236) Pub Date : 2014-07-08
    JinGyu Ock,Wei Li

    A microarray containing three-dimensional (3D) tissue models is a promising substitute for the two-dimensional (2D) cell-based microarrays currently available for high throughput, tissue-based biomedical assays. A cell culture microenvironment similar to in vivo conditions could be achieved with biodegradable porous scaffolds. In this study, a laser foaming technique is developed to create an array of micro-scale 3D porous scaffolds. The effects of major process parameters and the morphology of the resulting porous structure were investigated. For comparison, cell culture studies were conducted with both foamed and unfoamed samples using T98G cells. The results show that by laser foaming gas-impregnated polylactic acid it is possible to generate an array of inverse cone shaped wells with porous walls. The size of the foamed region can be controlled with laser power and exposure time, while the pore size of the scaffold can be manipulated with the saturation pressure. T98G cells grow well in the foamed scaffolds, forming clusters that have not been observed in 2D cell cultures. Cells are more viable in the 3D scaffolds than in the 2D cell culture cases. The 3D porous microarray could be used for parallel studies of drug toxicity, guided stem cell differentiation, and DNA binding profiles.

    更新日期:2019-11-01
  • One-stop microfiber spinning and fabrication of a fibrous cell-encapsulated scaffold on a single microfluidic platform.
    Biofabrication (IF 7.236) Pub Date : 2014-07-08
    D Y Park,C H Mun,E Kang,D Y No,J Ju,S H Lee

    This paper provides a method for microscale fiber spinning and the in situ construction of a 3D fibrous scaffold on a single microfluidic platform. This platform was also used to fabricate a variety of fibrous scaffolds with diverse compositions without the use of complicated devices. We explored the potential utility of the fibrous scaffolds for tissue engineering applications by constructing a fibrous scaffold encapsulating primary hepatocytes. The cells in scaffold were cultured over seven days and maintained higher viability comparing with 3D alginate non-fibrous block. The main advantage of this platform is that the fibrous structure used to form a scaffold can be generated without damaging the mechanically weak alginate fibers or encapsulated cells because all procedures are performed in a single platform without the intervention of the operator. In addition, the proposed fibrous scaffold permitted high diffusion capability of molecules, which enabled better viability of encapsulated cells than non-fibrous scaffold even in massive cell culture.

    更新日期:2019-11-01
  • Thermal inkjet bioprinting triggers the activation of the VEGF pathway in human microvascular endothelial cells in vitro.
    Biofabrication (IF 7.236) Pub Date : 2019-06-01
    Luis H Solis,Yoshira Ayala,Susana Portillo,Armando Varela-Ramirez,Renato Aguilera,Thomas Boland

    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.

    更新日期:2019-11-01
  • Printability of pulp derived crystal, fibril and blend nanocellulose-alginate bioinks for extrusion 3D bioprinting.
    Biofabrication (IF 7.236) Pub Date : 2019-02-12
    Zita M Jessop,Ayesha Al-Sabah,Neng Gao,Stuart Kyle,Bethan Thomas,Nafiseh Badiei,Karl Hawkins,Iain S Whitaker

    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 optimize and compare the printability of crystal, fibril and blend formulations of novel pulp derived nanocellulose bioinks and assess biocompatibility with human nasoseptal chondrocytes. 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 optimized 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 behavior 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 favorable rheological, swelling and biocompatibility properties of nanocellulose-alginate bioinks for extrusion-based bioprinting.

    更新日期:2019-11-01
  • Combined multi-nozzle deposition and freeze casting process to superimpose two porous networks for hierarchical three-dimensional microenvironment.
    Biofabrication (IF 7.236) Pub Date : 2014-01-17
    Jessica E Snyder,Philipp M Hunger,Chengyang Wang,Qudus Hamid,Ulrike G K Wegst,Wei Sun

    An engineered three-dimensional scaffold with hierarchical porosity and multiple niche microenvironments is produced using a combined multi-nozzle deposition-freeze casting technique. In this paper we present a process to fabricate a scaffold with improved interconnectivity and hierarchical porosity. The scaffold is produced using a two-stage manufacturing process which superimposes a printed porous alginate (Alg) network and a directionally frozen ceramic-polymer matrix. The combination of two processes, multi-nozzle deposition and freeze casting, provides engineering control of the microenvironment of the scaffolds over several length scales; including the addition of lateral porosity and the ratio of polymer to ceramic microstructures. The printed polymer scaffold is submerged in a ceramic-polymer slurry and subsequently, both structures are directionally frozen (freeze cast), superimposing and patterning both microenvironments into a single hierarchical architecture. An optional additional sintering step removes the organic material and densifies the ceramic phase to produce a well-defined network of open pores and a homogenous cell wall material composition. The techniques presented in this contribution address processing challenges, such as structure definition, reproducibility and fine adjustments of unique length scales, which one typically encounters when fabricating topological channels between longitudinal and transverse porous networks.

    更新日期:2019-11-01
  • Development of a microfabricated artificial limbus with micropockets for cell delivery to the cornea.
    Biofabrication (IF 7.236) Pub Date : 2013-04-18
    Ílida Ortega,Pallavi Deshpande,Andrew A Gill,Sheila MacNeil,Frederik Claeyssens

    The aim of this study was to develop a synthetic alternative to the human corneal limbus for use initially as an ex vivo model in which to study corneal stem cell function within a niche environment and ultimately to develop an implantable limbus for future clinical use. Microstereolithography was used for the fabrication of polyethylene glycol diacrylate (PEGDA) based rings on a macroscopic (1.2 cm) scale containing unique microfeatures (pockets) which were then modified with fibronectin to promote cell adhesion. These rings were designed to mimic the limbal area of the eye containing structures of the approximate size and shape of the stem cell microenvironments found in the palisades of Vogt. The attachment of rabbit limbal fibroblasts and rabbit limbal epithelial cells to the PEGDA rings was increased by pretreating the microfabricated structures with biotinylated fibronectin. Cell outgrowth from fibronectin coated microfabricated structures was 50% greater than from rings without structures or fibronectin coating. The cell loaded rings were then placed on an ex vivo wounded cornea model and the outgrowth of cells to form a multilayered epithelium was observed. We suggest this is a new approach to investigating limbal stem cells niches and the first steps towards a new approach for corneal regeneration.

    更新日期:2019-11-01
  • Tissue engineering of retina through high resolution 3-dimentional inkjet bioprinting.
    Biofabrication (IF 7.236) Pub Date : 2019-10-03
    Elahe Masaeli,Valérie Forster,Serge Picaud,Freshteh Karamali,Mohammad-Hossein Nasr-Esfahani,Christophe A Marquette

    The mammalian retina contains multiple cellular layers, each carrying out a specific task. Such a controlled organization should (Yue et al., 2015)be considered as a crucial factor for designing retinal therapies. The maintenance of retinal layered complexity through the use of scaffold-free techniques has recently emerged as a promising approach for clinical ocular tissue engineering. In an attempt to fabricate such layered retinal model, we are proposing herein a unique inkjet bioprinting system applied to the deposition of a photoreceptor cell (PRs) layer on top of a bioprinted retinal pigment epithelium (RPE), in a precise arrangement and without any carrier material. The results showed that, after bioprinting, both RPE and PRs were well positioned in a layered structure and expressed their structural markers, which was further demonstrated by ZO1, MITF, rhodopsin, opsin B, opsin R/G and PNA immunostaining, 3 days after bioprinting. We also showed that considerable amounts of human vascular endothelial growth factor (hVEGF) were released from the RPE printed layer, which confirmed formation of a functional RPE monolayer after bioprinting. Microstructures of bioprinted cells as well as phagocytosis of photoreceptor outer segments by apical RPE microvilli was finally established through transmission electron microscopy (TEM) imaging. In summary, using this carrier-free bioprinting method, it was possible to develop a reasonable in vitro retina model for studding some sight-threatening diseases such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP).

    更新日期:2019-11-01
  • Biomanufacturing of a novel in vitro biomimetic blood-brain barrier model.
    Biofabrication (IF 7.236) Pub Date : 2019-09-20
    Libiao Liu,Xinda Li,Xinzhi Zhang,Tao Xu

    Glioma is a malignant tumor that severely threatens human health. However, it is difficult for most therapeutic agents to penetrate through the blood-brain barrier (BBB) and exhibit their antineoplastic activity in the brain. In this manuscript, a biomimetic in vitro BBB model was created by a composite process, this model can provide a significant foundation for the research of drug transport, tumor treatment, tumor microenvironment and other fields. A series of tests and comparative experiments were performed to evaluate this model. The tests showed that the model enabled preliminary simulation of the structure and function of the BBB. Experimental results demonstrated: 1) the new technology enabled controlled release of growth factors and successfully induced endothelial progenitor cells into endothelial cells. Compared with the traditional gold standard, the Transwell model, the expression of four specific proteins that are related to the BBB characteristics was significantly increased (alkaline phosphatase(ALP) by 89.82%, γ-GT by 88.86%, zonula occludens-1 (ZO-1) by 57.40%, and Claudin-5 by 102.32%) in this model; 2) Astrocytes had a promoting effect on the microvascular endothelial cells to form tight junction (ZO-1 increased by 249.35%, Claudin-5 increased by 184.99%), and there was a great difference between whether these two types of cells were contact cultured or not; 3) The gelatinous cell U118 had a destructive effect on the tight junction of BBB (ZO-1 decreased by 55.86%, Claudin-5 decreased by 37.84%).

    更新日期:2019-11-01
  • 4D anisotropic skeletal muscle tissue constructs fabricated by staircase effect strategy.
    Biofabrication (IF 7.236) Pub Date : 2019-04-27
    Shida Miao,Margaret Nowicki,Haitao Cui,Se-Jun Lee,Xuan Zhou,David K Mills,Lijie Grace Zhang

    Like the morphology of native tissue fiber arrangement (such as skeletal muscle), unidirectional anisotropic scaffolds are highly desired as a means to guide cell behavior in anisotropic tissue engineering. In contrast, contour-like staircases exhibit directional topographical cues and are judged as an inevitable defect of fused deposition modeling (FDM). In this study, we will translate this staircase defect into an effective bioengineering strategy by integrating FDM with surface coating technique (FCT) to investigate the effect of topographical cues on regulating behaviors of human mesenchymal stem cells (hMSCs) toward skeletal muscle tissues. This integrated approach serves to fabricate shape-specific, multiple dimensional, anisotropic scaffolds using different biomaterials. 2D anisotropic scaffolds, first demonstrated with different polycaprolactone concentrations herein, efficiently direct hMSC alignment, especially when the scaffold is immobilized on a support ring. By surface coating the polymer solution inside FDM-printed sacrificial structures, 3D anisotropic scaffolds with thin wall features are developed and used to regulate seeded hMSCs through a self-established rotating bioreactor. Using layer-by-layer coating, along with a shape memory polymer, smart constructs exhibiting shape fix and recovery processes are prepared, bringing this study into the realm of 4D printing. Immunofluorescence staining and real-time quantitative polymerase chain reaction analysis confirm that the topographical cues created via FCT significantly enhance the expression of myogenic genes, including myoblast differentiation protein-1, desmin, and myosin heavy chain-2. We conclude that there are broad application potentials for this FCT strategy in tissue engineering as many tissues and organs, including skeletal muscle, possess highly organized and anisotropic extracellular matrix components.

    更新日期:2019-11-01
  • High resolution bioprinting of multi-component hydrogels.
    Biofabrication (IF 7.236) Pub Date : 2019-06-19
    Ralf Zimmermann,Christoph Hentschel,Felix Schrön,Denise Moedder,Teresa Büttner,Passant Atallah,Thomas Wegener,Thomas Gehring,Steffen Howitz,Uwe Freudenberg,Carsten Werner

    Materials capable of directing cell fate by providing spatially-graded mechanical and biomolecular cues are critically important in the reconstitution of living matter. Herein, we report a multi-component inkjet bioprinting method that allows for spatially varying composition and network properties in cell-instructive glycosaminoglycan (GAG)-based biohybrid and pure poly(ethylene glycol) hydrogels with unprecedented (50 μm) resolution. The principle relies on the covalent crosslinking of different polymeric precursors through a very rapid bio-orthogonal Michael type addition scheme adjusted in ways to occur during the fusion of bio-ink droplets prior to and upon contact with the target. Exemplary data show that chemotactic molecular gradients produced by this approach within printed GAG-gels of defined zonal architecture can effectively direct migratory activity and morphogenesis of embedded human bone-marrow derived mesenchymal stem cells. The introduced methodology is expected to enable a new, holistic level of control over reductionistic tissue and organoid models.

    更新日期:2019-11-01
  • Multi-cellular engineered living systems: building a community around responsible research on emergence.
    Biofabrication (IF 7.236) Pub Date : 2019-06-04
    Matthew Sample,Marion Boulicault,Caley Allen,Rashid Bashir,Insoo Hyun,Megan Levis,Caroline Lowenthal,David Mertz,Nuria Montserrat,Megan J Palmer,Krishanu Saha,Jeremiah Zartman

    Ranging from miniaturized biological robots to organoids, multi-cellular engineered living systems (M-CELS) pose complex ethical and societal challenges. Some of these challenges, such as how to best distribute risks and benefits, are likely to arise in the development of any new technology. Other challenges arise specifically because of the particular characteristics of M-CELS. For example, as an engineered living system becomes increasingly complex, it may provoke societal debate about its moral considerability, perhaps necessitating protection from harm or recognition of positive moral and legal rights, particularly if derived from cells of human origin. The use of emergence-based principles in M-CELS development may also create unique challenges, making the technology difficult to fully control or predict in the laboratory as well as in applied medical or environmental settings. In response to these challenges, we argue that the M-CELS community has an obligation to systematically address the ethical and societal aspects of research and to seek input from and accountability to a broad range of stakeholders and publics. As a newly developing field, M-CELS has a significant opportunity to integrate ethically responsible norms and standards into its research and development practices from the start. With the aim of seizing this opportunity, we identify two general kinds of salient ethical issues arising from M-CELS research, and then present a set of commitments to and strategies for addressing these issues. If adopted, these commitments and strategies would help define M-CELS as not only an innovative field, but also as a model for responsible research and engineering.

    更新日期:2019-11-01
  • In situ prevascularization designed by laser-assisted bioprinting: effect on bone regeneration.
    Biofabrication (IF 7.236) Pub Date : 2019-06-01
    Olivia Kérourédan,Davit Hakobyan,Murielle Rémy,Sophia Ziane,Nathalie Dusserre,Jean-Christophe Fricain,Samantha Delmond,Noëlie B Thébaud,Raphaël Devillard

    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.

    更新日期:2019-11-01
  • 3D bioprinting of hydrogel constructs with cell and material gradients for the regeneration of full-thickness chondral defect using a microfluidic printing head.
    Biofabrication (IF 7.236) Pub Date : 2019-06-01
    Joanna Idaszek,Marco Costantini,Tommy A Karlsen,Jakub Jaroszewicz,Cristina Colosi,Stefano Testa,Ersilia Fornetti,Sergio Bernardini,Martyna Seta,Kaja Kasarełło,Robert Wrzesień,Stefano Cannata,Andrea Barbetta,Cesare Gargioli,Jan E Brinchman,Wojciech Święszkowski

    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.

    更新日期:2019-11-01
  • Fabrication of elastomer pillar arrays with elasticity gradient for cell migration, elongation and patterning.
    Biofabrication (IF 7.236) Pub Date : 2019-05-16
    Bin Wang,Jian Shi,Jin Wei,Xiaolong Tu,Yong Chen

    The elasticity of the cell and that of the supporting extracellular matrices (ECMs) in tissue are correlated. In some cases, the modulus of the ECM varies with a high spatial gradient. To study the effect of such a modulus gradient on the cell culture behavior, we proposed a novel yet straightforward method to fabricate elastomeric micropillar substrates with different height gradients, which could provide a large range of elasticity gradient from 2.4 kPa to 60 kPa. The micropillars were integrated into a microfluidic chip to demonstrate the elasticity variation, with the theoretical results proving that the elasticity of the two micropillar substrates was in the same range but with distinguished gradient strengths. Fibroblast seeded on the micropillar substrates showed migration toward the stiffer area but their elongation highly depended on the strength of the elasticity gradient. In the case of high gradient strength, cells could easily migrate to the stiffer area and then elongated perpendicularly to their migration direction. Otherwise, cells were mostly elongated in the direction of the gradient. Our results also showed that when the cell density was sufficiently high, cells tended to be oriented in the same direction locally, which was affected by both underneath pillars and cell-cell contact. The elasticity gradients could also be generated in a ripple shape, and the cell behavior showed the feasibility of using the micropillars for cell patterning applications. Moreover, the gradient pillar substrates were further used for the aggregate formation of induced pluripotent stem cells, thus providing an alternative substrate to study the effect of substrate elasticity on stem cell behavior and differentiation.

    更新日期:2019-11-01
  • A 3D construct of the intestinal canal with wrinkle morphology on a centrifugation configuring microfluidic chip.
    Biofabrication (IF 7.236) Pub Date : 2019-05-16
    Yu Wang,Zixing Shao,Wenchen Zheng,Yuanyuan Xie,Guoan Luo,Mingyu Ding,Qionglin Liang

    A new in vitro gut microfluidic chip that mimics in vivo intestinal canal morphology and stimulation is developed to contribute to research into tissue engineering, and intestinal development and function. This strategy utilizes centrifugation to configure spatial cells along the side wall of a vertical cylinder-like microfluidic chamber, by which a tubular intestinal epithelium cell sheet is formed. Diverse intestinal cell lines are inoculated to address this approach. Furthermore, to generate microenvironmental stimulation, low-level centrifugation introduces fluid flow to this microfluidic system perpendicularly acting on cell sheet cultivation for several days. Fluid flow engenders the sectional cell sheet to bend toward the cell chamber lumen, which manifests an intestinal epithelium vaulted and wrinkle morphology. This may mimic the fluid flow existing in in vivo material transportation and the absorption of the gut epithelium barrier. In addition, the same fluid flow stimulation was reproduced in another Transwell system, which also exhibited a wrinkle epithelium cell sheet. Under fluid flow stimulation, some of the villus specific genes' expression level increased in the microfluidics and Transwell insert. Thus, this new centrifugation configuring gut microfluidic chip may offer novel insights into the research of intestinal structure and function.

    更新日期:2019-11-01
  • Achieving molecular orientation in thermally extruded 3D printed objects.
    Biofabrication (IF 7.236) Pub Date : 2019-04-27
    Salim A Ghodbane,N Sanjeeva Murthy,Michael G Dunn,J Kohn

    Three-dimensional (3D) printing is used to fabricate tissue scaffolds. Polymer chains in these objects are typically unoriented. The mechanical properties of these scaffolds can be significantly enhanced by proper alignment of polymer chains. However, post-processing routes to increase orientation can be limited by the geometry of the printed object. Here, we show that it is possible to orient polymer chains during printing by optimizing printing parameters to take advantage of the flow characteristics of the polymer. This is demonstrated by printing a polymeric scaffold for meniscus regeneration using poly(desaminotyrosyl-tyrosine dodecyl dodecanedioate), poly(DTD DD). Alignment of polymer chains was achieved by translating the printhead at sufficiently high speeds when the polymer was still in a semi-solid state as it cooled from the fluid state at the tip of the nozzle using a critical combination of nozzle diameter, extrusion pressure, and temperature. The degree of orientation as evaluated by x-ray diffraction and thermal shrinkage, was greater than that of drawn fibers. Significant orientation and defect-free printing was achieved even for scaffolds with complex geometries. The ability to orient polymers during 3D printing has the potential to combine the advantages of 3D printing with the superior mechanical performance of more conventional polymer processing methods, such as drawing.

    更新日期:2019-11-01
  • Multi-level customized 3D printing for autogenous implants in skull tissue engineering.
    Biofabrication (IF 7.236) Pub Date : 2019-03-28
    Hongqing Chen,Jing Zhang,Xinda Li,Libiao Liu,Xinzhi Zhang,Dongni Ren,Cheng Ma,Lei Zhang,Zhou Fei,Tao Xu

    Three-dimensional (3D) printing of decellularized extracellular matrix (ECM) has been achieved to ensure real physiological environments for tissue engineering. However, the limited source, biocompatibility, and biosafety of decellularized ECM are deficiencies in its large clinical use. Autogenous ECM is biocompatible, bioactive, and biosafe, making it an optimal choice for future clinical applications of 3D printing. Here, we developed a multi-level customized 3D printing (MLC-3DP) strategy applying autogenous bone matrix (Auto-BM). This MLC-3DP includes shape specificity (shape), material specificity (Auto-BM), and cell specificity (autogenous cells) for true patient-specific repairs. Auto-BM (skull flaps) is readily accessible for specific patients after craniectomy, providing sufficient autogenous materials for MLC-3DP. Under mild conditions of this strategy, human-scale 3D printed samples can be fabricated using bioactive micron-sized Auto-BM particles. Multi-level customized autogenous bones (MLC-Auto-Bones) are finally obtained by combining autogenous bone marrow-derived mesenchymal stem cells (Auto-BMSCs). With autogenous materials and cells, MLC-Auto-Bones are inherently biocompatible and biosafe, providing good bioactivity for osteogenesis. In this implant, Auto-BMSCs can spontaneously differentiate into osteoblasts in vitro without additional osteogenic factors. In critical-sized skull defect models in vivo (3 months), implants integrate tightly to the defects' margin, facilitate mineralization, and generate vascularized mature bone. This work provides not only feasibility for patient-specific implants for skull defects, but also potential patient-specific solutions for other similar clinical requirements.

    更新日期:2019-11-01
  • Self-organization of hepatocyte morphogenesis depending on the size of collagen microbeads relative to hepatocytes.
    Biofabrication (IF 7.236) Pub Date : 2019-04-27
    Mohammad Ajoudanian,Keita Enomoto,Yasuaki Tokunaga,Hiroshi Minami,Seok Chung,Kazuo Tanishita,Roger D Kamm,Ryo Sudo

    Recent advances in microfabrication technologies have enabled us to construct collagen gel microbeads, which can be cultured with hepatocytes. However, little is known about the hepatocyte-collagen gel microbead interactions. Here, we aimed to clarify the effects of the balance between cell-cell and cell-collagen gel microbead interactions on hepatocyte morphogenesis and functions. The magnitude of cell-microbead interactions was controlled by changing the size of the microbeads, which were smaller than, comparable to, and larger than hepatocytes. These small, medium, and large microbeads were cultured separately with primary hepatocytes. Phase-contrast and time-lapse imaging revealed that the medium microbeads significantly induced the construction of 3D structures composed of the microbeads and hepatocytes in a self-organizing manner, whereas hepatocytes formed 2D monolayers with the small or large microbeads. These results suggest that only the medium microbeads induced the 3D tissue formation of hepatocytes. Furthermore, liver-specific functions, such as albumin secretion and ammonia clearance, were significantly upregulated in the 3D structures. These findings are critical to understand how to control the construction of 3D hepatocyte tissues with hydrogel microbeads in the context of biofabrication.

    更新日期:2019-11-01
  • 3D bioprinting of heterogeneous bi- and tri-layered hollow channels within gel scaffolds using scalable multi-axial microfluidic extrusion nozzle.
    Biofabrication (IF 7.236) Pub Date : 2018-12-12
    Rana Attalla,Erin Puersten,Nidhi Jain,P Ravi Selvaganapathy

    One of the primary focuses in recent years in tissue engineering has been the fabrication and integration of vascular structures into artificial tissue constructs. However, most available methodologies lack the ability to create multi-layered concentric conduits inside natural extracellular matrices (ECMs) and gels that replicate more accurately the hierarchical architecture of biological blood vessels. In this work, we present a new microfluidic nozzle design capable of multi-axial extrusion in order to 3D print and pattern bi- and tri-layered hollow channel structures. This nozzle allows, for the first time, for these structures to be embedded within layers of gels and ECMs in a fast, simple and low-cost manner. By varying flow rates (1-6 ml min-1), printspeeds (1-16 m min-1), and material concentration (25-175 mM and 1.5%-2.5% for calcium chloride and alginate, respectively) we are able to accurately determine the operational printing range as well as achieve a wide range of conduit dimensions (0.69-2.31 mm) that can be printed within a few seconds. Our scalable design allows for multi-axial extrusion and versatility in material incorporation in order to create heterogeneous structures. We demonstrate the ability to print distinct concentric layers of different cell types, namely endothelial cells and fibroblasts. By incorporating various layers of different cell-friendly materials (such as collagen and fibrin) alongside materials with high mechanical strength (i.e. alginate), we were able to increase long-term cell viability and growth without compromising the structural integrity. In this way, we can improve cellular adhesion in our biocompatible constructs as well as allow them to remain structurally sound. We are able to realize complex heterogeneous, hierarchical architectures that have strong potential for use not only in vascular tissue applications, but also in other artificially fabricated tubular or fiber-like structures such as skeletal muscle or nerve conduits.

    更新日期:2019-11-01
  • Homogeneous hydroxyapatite/alginate composite hydrogel promotes calcified cartilage matrix deposition with potential for three-dimensional bioprinting.
    Biofabrication (IF 7.236) Pub Date : 2018-12-14
    Fu You,Xiongbiao Chen,D M L Cooper,Tuanjie Chang,B Frank Eames

    Calcified cartilage regeneration plays an important role in successful osteochondral repair, since it provides a biological and mechanical transition from the unmineralized cartilage at the articulating surface to the underlying mineralized bone. To biomimic native calcified cartilage in engineered constructs, here we test the hypothesis that hydroxyapatite (HAP) stimulates chondrocytes to secrete the characteristic matrix of calcified cartilage. Sodium citrate (SC) was added as a dispersant of HAP within alginate (ALG), and homogeneous dispersal of HAP within ALG hydrogel was confirmed using sedimentation tests, electron microscopy, and energy dispersive spectroscopy. To examine the biological performance of ALG/HAP composites, chondrocyte survival and proliferation, extracellular matrix production, and mineralization potential were evaluated in the presence or absence of the HAP phase. Chondrocytes in ALG/HAP constructs survived well and proliferated, but also expressed higher levels of calcified cartilage markers compared to controls, including Collagen type X secretion, alkaline phosphatase (ALP) activity, and mineral deposition. Compared to controls, ALG/HAP constructs also showed an elevated level of mineralized matrix in vivo when implanted subcutaneously in mice. The printability of ALG/HAP composite hydrogel precursors was verified by 3D printing of ALG/HAP hydrogel scaffolds with a porous structure. In summary, these results confirm the hypothesis that HAP in ALG hydrogel stimulates chondrocytes to secrete calcified matrix in vitro and in vivo and reveal that ALG/HAP composites have the potential for 3D bioprinting and osteochondral regeneration.

    更新日期:2019-11-01
  • Oxygen transporter for the hypoxic transplantation site.
    Biofabrication (IF 7.236) Pub Date : 2018-12-14
    Hirotake Komatsu,Colin A Cook,Nelson Gonzalez,Leonard Medrano,Mayra Salgado,Feng Sui,Junfeng Li,Fouad Kandeel,Yoko Mullen,Yu-Chong Tai

    Cell transplantation is a promising treatment for complementing lost function by replacing new cells with a desired function, e.g. pancreatic islet transplantation for diabetics. To prevent cell obliteration, oxygen supply is critical after transplantation, especially until the graft is sufficiently re-vascularized. To supply oxygen during this period, we developed a chemical-/electrical-free implantable oxygen transporter that delivers oxygen to the hypoxic graft site from ambient air by diffusion potential. This device is simply structured using a biocompatible silicone-based body that holds islets, connected to a tube that opens outside the body. In computational simulations, the oxygen transporter increased the oxygen level to >120 mmHg within grafts; in contrast, a control device that did not transport oxygen showed <6.5 mmHg. In vitro experiments demonstrated similar results. To test the effectiveness of the oxygen transporter in vivo, we transplanted pancreatic islets, which are susceptible to hypoxia, subcutaneously into diabetic rats. Islets transplanted using the oxygen transporter showed improved graft viability and cellular function over the control device. These results indicate that our oxygen transporter, which is safe and easily fabricated, effectively supplies oxygen locally. Such a device would be suitable for multiple clinical applications, including cell transplantations that require changing a hypoxic microenvironment into an oxygen-rich site.

    更新日期:2019-11-01
  • Reactive jet impingement bioprinting of high cell density gels for bone microtissue fabrication.
    Biofabrication (IF 7.236) Pub Date : 2018-12-14
    Ricardo da Conceicao Ribeiro,Deepali Pal,Ana Marina Ferreira,Piergiorgio Gentile,Matthew Benning,Kenneth Dalgarno

    Advances in three-dimensional cell cultures offer new opportunities in biomedical research and drug development. However, there are still challenges to overcome, including the lack of reliability, repeatability and complexity of tissues obtained by these techniques. In this study, we describe a new bioprinting system called reactive jet impingement (ReJI) for the bioprinting of cell-laden hydrogels. Droplets of gel precursor solutions are jetted at one another such that they meet and react in mid-air before the gel droplets fall to the substrate. This technique offers a combination of deposition rate, cell density and cell viability which is not currently matched by any other bioprinting technique. The importance of cell density is demonstrated in the development of bone microtissues derived from immortalised human bone marrow stem cells. The cells were printed with high viability within a collagen-alginate-fibrin gel, and tissue specific gene expression shows significantly higher tissue maturation rates using the ability of the ReJI system to deposit gels with a high cell density.

    更新日期:2019-11-01
  • Fabrication of modular hyaluronan-PEG hydrogels to support 3D cultures of hepatocytes in a perfused liver-on-a-chip device.
    Biofabrication (IF 7.236) Pub Date : 2018-12-14
    Jonas Christoffersson,Christopher Aronsson,Michael Jury,Robert Selegård,Daniel Aili,Carl-Fredrik Mandenius

    Liver cell culture models are attractive in both tissue engineering and for development of assays for drug toxicology research. To retain liver specific cell functions, the use of adequate cell types and culture conditions, such as a 3D orientation of the cells and a proper supply of nutrients and oxygen, are critical. In this article, we show how extracellular matrix mimetic hydrogels can support hepatocyte viability and functionality in a perfused liver-on-a-chip device. A modular hydrogel system based on hyaluronan and poly(ethylene glycol) (HA-PEG), modified with cyclooctyne moieties for bioorthogonal strain-promoted alkyne-azide 1, 3-dipolar cycloaddition (SPAAC), was developed, characterized, and compared for cell compatibility to hydrogels based on agarose and alginate. Hepatoma cells (HepG2) formed spheroids with viable cells in all hydrogels with the highest expression of albumin and urea in alginate hydrogels. By including an excess of cyclooctyne in the HA backbone, azide-modified cell adhesion motifs (linear and cyclic RGD peptides) could be introduced in order to enhance viability and functionality of human induced pluripotent stem cell derived hepatocytes (hiPS-HEPs). In the HA-PEG hydrogels modified with cyclic RGD peptides hiPS-HEPs migrated and grew in 3D and showed an increased viability and higher albumin production compared to when cultured in the other hydrogels. This flexible SPAAC crosslinked hydrogel system enabled fabrication of perfused 3D cell culture of hiPS-HEPs and is a promising material for further development and optimization of liver-on-a-chip devices.

    更新日期:2019-11-01
  • Fabrication of perfusable 3D hepatic lobule-like constructs through assembly of multiple cell type laden hydrogel microstructures.
    Biofabrication (IF 7.236) Pub Date : 2018-12-14
    Juan Cui,Huaping Wang,Zhiqiang Zheng,Qing Shi,Tao Sun,Qiang Huang,Toshio Fukuda

    The in vitro reproduction of three-dimensional (3D) cellular constructs to physiologically mimic human liver is highly desired for drug screening and clinical research. However, the fabrication of a liver-mimetic 3D model using traditional bottom-up technologies is challenging owing to the complex architecture and specific functions of real liver tissue. This work proposes a versatile strategy for spatially assembling gear-like microstructures encapsulating multiple cell types, and reorganizing them into 3D lobule-like micro-architecture with physiological relevance to native liver tissue. Gear-like microstructures were fabricated by photo-crosslinking poly(ethylene glycol) diacrylate (PEGDA) hydrogel mixed with hepatocytes and fibroblasts, in a digital micromirror device (DMD)-based microfluidic channel. The microstructures were assembled through coordinated micromanipulation based on local fluid force, and spatially self-aligned through hydrophilic-hydrophobic interactions into a 3D integrated construct with lobule-like morphology and a perfusable central lumen. The resulting 3D lobule-like constructs allowed long-term co-culture of hepatocytes and fibroblasts with high cell viability. The co-cultured constructs enhanced hepatocyte proliferation and spreading, as well as liver functions including a 50% increase in albumin secretion and urea synthesis. For hepatotoxicity assessment, the 3D lobule-like construct enabled drug perfusion through its built-in lumen for simulation of drug diffusion in the liver, which could improve the response sensitivity and efficiency to hepatotoxic drug. These results demonstrated that this method provides a valuable 3D co-culture model with perfusable lobule-like architecture and physiological functions, which has potential applications in drug discovery and tissue engineering applications.

    更新日期:2019-11-01
  • Double-layer perfusable collagen microtube device for heterogeneous cell culture.
    Biofabrication (IF 7.236) Pub Date : 2018-12-01
    Shun Itai,Hisatsugu Tajima,Hiroaki Onoe

    In vitro perfusable 3D tissue models mimic in vivo tissues and have several benefits in drug testing. However, processes used to fabricate these models often tend to be complicated. Here, we present a double-layer perfusable collagen tube device for multilayered in vitro 3D cell culture. The device is simply made by the repetition of a molding process. The thicknesses of the collagen layers in the tube device can be flexibly designed, and heterogeneous cell types can be co-cultured in/on each collagen layer. Moreover, while our collagen tube is directly attached to silicone tubes, the collagen tube can easily be connected to an external pump system for perfusion culture. We fabricated six different sizes of collagen devices (inner diameter approximately 300-1000 μm) using different molds, and successfully controlled the coefficients of variation to be below 5% for the diameters of each layer for all six device sizes. The device is strong enough to manipulate with tweezers, and can remain stable for more than 3 months in a medium. For the cell culture, we successfully and correctly encapsulated cells in the layer shape at the desired position, and confirmed cell migration. Using the perfusion culture, we demonstrated that the alignments of the HUVEC actin filaments become parallel to the flow direction. We believe that our device could advance the easy fabrication of various tissue models (that is, models mimicking in vivo tissues). In particular, the device could help fabricate vascularized tissue models, and contribute to the development of pharmacokinetic testing platforms and regenerative medicine.

    更新日期:2019-11-01
  • Porous tissue strands: avascular building blocks for scalable tissue fabrication.
    Biofabrication (IF 7.236) Pub Date : 2018-11-24
    Yang Wu,Monika Hospodiuk,Weijie Peng,Hemanth Gudapati,Thomas Neuberger,Srinivas Koduru,Dino J Ravnic,Ibrahim T Ozbolat

    The scalability of cell aggregates such as spheroids, strands, and rings has been restricted by diffusion of nutrient and oxygen into their core. In this study, we introduce a novel concept in generating tissue building blocks with micropores, which represents an alternative solution for vascularization. Sodium alginate porogens were mixed with human adipose-derived stem cells, and loaded into tubular alginate capsules, followed by de-crosslinking of the capsules. The resultant cellular structure exhibited a porous morphology and formed cell aggregates in the form of strands, called 'porous tissue strands (pTSs).' Three-dimensional reconstructions show that pTSs were able to maintain ∼25% porosity with a high pore interconnectivity (∼85%) for 3 weeks. Owing to the porous structure, pTSs showed up-regulated cell viability and proliferation rate as compared to solid counterparts throughout the culture period. pTSs also demonstrated self-assembly capability through tissue fusion yielding larger-scale patches. In this paper, chondrogenesis and osteogenesis of pTSs were also demonstrated, where the porous microstructure up-regulated both chondrogenic and osteogenic functionalities indicated by cartilage- and bone-specific immunostaining, quantitative biochemical assessment and gene expression. These findings indicated the functionality of pTSs, which possessed controllable porosity and self-assembly capability, and had great potential to be utilized as tissue building blocks in distinct applications such as cartilage and bone regeneration.

    更新日期:2019-11-01
  • Special issue on bioinks.
    Biofabrication (IF 7.236) Pub Date : 2018-11-24
    Jürgen Groll,James J Yoo

    更新日期:2019-11-01
  • A definition of bioinks and their distinction from biomaterial inks.
    Biofabrication (IF 7.236) Pub Date : 2018-11-24
    J Groll,J A Burdick,D-W Cho,B Derby,M Gelinsky,S C Heilshorn,T Jüngst,J Malda,V A Mironov,K Nakayama,A Ovsianikov,W Sun,S Takeuchi,J J Yoo,T B F Woodfield

    Biofabrication aims to fabricate biologically functional products through bioprinting or bioassembly (Groll et al 2016 Biofabrication 8 013001). In biofabrication processes, cells are positioned at defined coordinates in three-dimensional space using automated and computer controlled techniques (Moroni et al 2018 Trends Biotechnol. 36 384-402), usually with the aid of biomaterials that are either (i) directly processed with the cells as suspensions/dispersions, (ii) deposited simultaneously in a separate printing process, or (iii) used as a transient support material. Materials that are suited for biofabrication are often referred to as bioinks and have become an important area of research within the field. In view of this special issue on bioinks, we aim herein to briefly summarize the historic evolution of this term within the field of biofabrication. Furthermore, we propose a simple but general definition of bioinks, and clarify its distinction from biomaterial inks.

    更新日期:2019-11-01
  • Perfusable and stretchable 3D culture system for skin-equivalent.
    Biofabrication (IF 7.236) Pub Date : 2018-11-16
    Nobuhito Mori,Yuya Morimoto,Shoji Takeuchi

    This study describes a perfusable and stretchable culture system for a skin-equivalent. The system is comprised of a flexible culture device equipped with connections that fix vascular channels of the skin-equivalent and functions as an interface for an external pump. Furthermore, a stretching apparatus for the culture device can be fabricated using rapid prototyping technologies, which allows for easy modifications of stretching parameters. When cultured under dynamically stretching and perfusion conditions, the skin-equivalent exhibits improved morphology. The epidermal layer becomes thicker and more differentiated than that cultured without the stretching stimuli or under statically-stretched conditions, and the dermal layer was more densely populated with dermal fibroblasts than that cultured without perfusion due to the nutrient and oxygen supply by perfusion via the vascular channels. Therefore, the system is useful for the improvement and biological studies of skin-equivalents.

    更新日期:2019-11-01
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