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Recent advances in three-dimensional (3-D) printing offer an excellent opportunity to address critical challenges faced by current tissue engineering approaches. Alginate hydrogels have been used extensively as bioinks for 3-D bioprinting. However, most previous research has focused on native alginates with limited degradation. The application of oxidized alginates with controlled degradation in bioprinting has not been explored. Here, a collection of 30 different alginate hydrogels with varied oxidation percentages and concentrations was prepared to develop a bioink platform that can be applied to a multitude of tissue engineering applications. The authors systematically investigated the effects of two key material properties (i.e. viscosity and density) of alginate solutions on their printabilities to identify a suitable range of material properties of alginates to be applied to bioprinting. Further, four alginate solutions with varied biodegradability were printed with human adipose-derived stem cells (hADSCs) into lattice-structured, cell-laden hydrogels with high accuracy. Notably, these alginate-based bioinks were shown to be capable of modulating proliferation and spreading of hADSCs without affecting the structure integrity of the lattice structures (except the highly degradable one) after 8days in culture. This research lays a foundation for the development of alginate-based bioink for tissue-specific tissue engineering applications.

A bioimpedance platform is presented as a promising tool for non-invasive real-time monitoring of the entire process of three-dimensional (3D) cell culturing in a hydrogel scaffold. In this study, the dynamics involved in the whole process of 3D cell culturing, starting from polymerisation of a bare 3D gelatin scaffold, to human mesenchymal stem cell (MSC) encapsulation and proliferation, was monitored over time. The platform consists of a large rectangular culture chamber with four embedded vertical gold plate electrodes that were exploited in two- and three terminal (2T and 3T) measurement configurations. By switching between the different combinations of electrode couples, it was possible to generate a multiplexing-like approach, which allowed for collecting spatially distributed information within the 3D space. Computational finite element (FE) analysis and electrochemical impedance spectroscopic (EIS) characterisation were used to determine the configurations' sensitivity field localisation. The 2T setup gives insight into the interfacial phenomena at both electrode surfaces and covers the central part of the 3D cell culture volume, while the four 3T modes provide focus on the dynamics at the corners of the 3D culture chamber. By combining a number of electrode configurations, complementary spatially distributed information on a large 3D cell culture can be obtained with maximised sensitivity in the entire 3D space. The experimental results show that cell proliferation can be monitored within the tested biomimetic environment, paving the way to further developments in bioimpedance tracking of 3D cell cultures and tissue engineering.

The success of stem cell therapies is highly dependent on the ability to control their programmed differentiation. So far, it is commonly believed that the differentiation behavior of stem cells is supposed to be identical when they are cultured on the same homogeneous platform. However, in this report, we show that this is not always true. By utilizing a double-ion-triggered shape memory effect, the pre-seeded hMSCs were controllably located in different growth positions. Here, we demonstrate for the first time that the differentiation behavior of hMSCs is highly sensitive to their growth position on a hydrogel scaffold. This work will not only enrich the mechanisms for controlling the differentiation of stem cells, but also offer a one-of-a-kind platform to achieve a heterogeneously differentiated stem cell-seeded hydrogel scaffold for complex biological applications.

A novel and modular strategy has been developed for the preparation of reactive and functionalized hydrogels. In this strategy, thiol–epoxy coupling chemistry was employed for the formation of a hydrophilic network. The hydroxyl groups, generated during the coupling process, were then engaged in anchoring a fluorescent probe to the hydrogel scaffold.

Background: Early laboratory detection of Mycobacterium tuberculosis is crucial for controlling tuberculosis. We developed a hydrogel mycobacterial culture method that retains the advantages of both solid and liquid methods in terms of speed, cost, and efficiency. Methods: Mycobacterium bovis bacillus Calmette-Guerin (BCG) suspensions and 200 acid-fast bacilli (AFB)-positive clinical specimens were inoculated in Middlebrook 7H9 liquid media (Becton-Dickinson and Company, USA) and mixed with 75 μL of 9-fluorenylmethoxycarbonyl (Fmoc)-Phe-Phe-OH hydrogel stock solution in an Eppendorf tube just before culture incubation. The mixtures were cultured at 37°C for as long as 14 days to monitor culture status. Results: The number of M. bovis BCG increased with time. For 200 AFB smear-positive specimens, 155 of 158 conventional culture-positive specimens and 4 culture-negative or contaminated specimens yielded positive cultures within 14 days. For 128 specimens positive with the liquid culture method, the time to positive culture using the hydrogel method (mean, 12.6 days; range, 7 to 14 days) was significantly shorter than that for conventional liquid culture (mean, 16.2 days; range, 6 to 31 days; P

Medtronic's INFUSE Bone Graft provides surgeons with a potent tool for stimulating bone formation. Current delivery vehicles that rely on Absorbable Collagen Sponges (ACS) require excessive quantities of the active ingredient in INFUSE, recombinant human Bone Morphogenic Protein-2 (rhBMP2), to achieve physiologically relevant concentrations of the growth factor, driving up the cost of the product and increasing the likelihood of undesirable side effects in neighboring tissues. We demonstrate that a simple light-mediated, thiol-ene chemistry can be used to create an effective polymer delivery vehicle for rhBMP2, eliminating the use of xenographic materials and reducing the dose of rhBMP2 required to achieve therapeutic effects. Comprised entirely of synthetic components, this system entraps rhBMP2 within a biocompatible hydrogel scaffold that is degraded by naturally occurring remodeling enzymes, clearing the way for new tissue formation. When tested side-by-side with ACS in a critical-sized bone defect model in rats, this polymeric delivery system significantly increased bone formation over ACS controls.

Regeneration of skin after a large area wound or burn is often difficult without the aid of a scaffold. In this work we developed a 3D macroporous chitin/poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) hydrogel blend by normal solution chemistry and lyophilized it to get the scaffold. The developed scaffold was then characterized and it showed a slow degrading as well as biocompatible nature. The blend hydrogel scaffold showed 67% porosity and the pore size was less than 20 μm. Cytocompatibility of the scaffold was proved by Alamar blue assay using Human Dermal Fibroblast cells (HDF). The blend hydrogel scaffold showed a two fold increase in cell number over control within three days of culture. The chitin/PHBV blend also showed enhanced HDF cell attachment and proliferation. These preliminary results prove its suitability for scaffold based skin tissue regeneration.

Cell–cell interactions are critical to understanding functional tissues. A number of stem cell populations have been shown to receive key regulatory information from endothelial cells (ECs); however, the role of ECs in the retinal stem and progenitor cell (RSPC) niche has been largely unexplored. To gain greater insight into the role of ECs on RSPC fate, a three-dimensional (3D) co-culture model, incorporating cell–cell interactions, was designed by covalently-modifying agarose hydrogels with growth factors and cell-adhesive peptides in defined volumes. Therein ECs adopted tubular-like morphologies similar to those observed in vivo , but not observed in two-dimensional (2D) cultures. Unexpectedly, ECs inhibited proliferation and differentiation of RSPCs, revealing, for the first time, the possible role of ECs on RSPC fate. This 3D hydrogel scaffold provides a simple, reproducible and versatile method with which to answer biological questions related to the cellular microenvironment.

Hydrogels are attractive materials for use as tissue engineering scaffolds [1]. Natural hydrogels, such as collagen, are both cytocompatible and highly biofunctional. However, they have somewhat constrained material properties and have an inherently large variability in composition due to their biological origin, making them more difficult to work with from an engineering viewpoint[2–4]. Here, we aim to use type 1 collagen — the most abundant protein in the body that maintains excellent cytocompatibility and can self assemble into a fibrillar network — as a base component for a photocrosslinkable biomaterial. One main advantage of this system over previous studies attempting to photocrosslink collagen is that the collagen retains its ability to self assemble, which provides a stable environment into which localized modifications can be made to the stiffness, porosity, and biochemical properties of the hydrogel scaffold. By taking advantage of the spatial control provided by the system, we can create complex 3-dimensional hydrogel scaffolds that have non-homogenous microenvironments.Copyright © 2011 by ASME

Vascularization for sufficient supplies of oxygen/nutrients and removal of waste in thicker tissue is one of the major challenges in tissue engineering. Here, we used three-dimensional bio-printing technology to engineer a vascular structure within hydrogel scaffold. Through layer-by-layer approach, we seeded endothelial cells in tubular form, which is embedded within three-dimensional collagen scaffold. Collagen hydrogel precursor was printed and polymerized. Endothelial cells in heated gelatin hydrogel were printed within the collagen scaffold, subsequently gelatin was liquefied and washed out in order to fabricate a fluidic channel with endothelial cell lining. The channels were connected to perfusion system. Specially-designed flow chamber was used for stable perfusion. The endothelial cells survived under flow condition, showing high viability and integration along the channel wall. The channel had a width 400–700 um, and was able to resist 200mmHg of pressure. Functional assessments of the printed vascular channels are in progress. We have demonstrated the capability of using three-dimensional bio-printing technology in rapid, simple, and highthroughput vasculature formation. The process has a potential application in vascular tissue engineering and study in angiogenesis.

Here we describe a simple and efficient method for fabricating natural and synthetic hydrogels into 3-D geometries with high aspect ratio and curvature. Fabricating soft hydrogels into such shapes using conventional techniques has been extremely difficult. Combination of laser ablation and sacrificial molding technique using calcium alginate minimizes the stress associated with separating the mold from the hydrogel structure, and therefore allows fabrication of complex structures without damaging them. As a demonstration of this technique, we have fabricated a microscale collagen structure mimicking the actual density and size of human intestinal villi. Colon carcinoma cell line, Caco-2 cells, was seeded onto the structure and cultured for 3 weeks until the whole structure was covered, forming finger-like structures mimicking the intestinal villi covered with epithelial cells. This method will enable construction of in vitro tissue models with physiologically realistic geometries at microscale resolutions.

Vascularization for adequate supplies of oxygen and nutrients is one of the challenges in tissue engineering. We report the three-dimensional cell printing technology to construct vascular structure in hydrogel scaffold which consist of endothelial cells and collagen scaffold. We constructed multi-layered, inter-connected channels within collagen hydrogel through layer-by-layer approach. Collagen hydrogel precursor was printed and cross-linked with a nebulized NaHCO 3 . Heated gelatin hydrogel was printed within the collagen scaffold as a sacrificial material, liquefied and washed out to fabricate channels after complete gelation of collagen scaffold. The channels was perfused by syringe pump showing ability to resist 200mmHg of trans-luminal pressure. Endothelial cells which form inner lining of the vascular system was printed within collagen precursor in tubular pattern. The capability of three-dimensional cell printing technology in vasculature formation without multiple complicated processes has a potential in vascular tissue engineering. The simplicity of the technology allows it to readily incorporate other types of cells or scaffold materials to construct complex tissue structure in the future.

One of the challenges in tissue engineering is to provide adequate supplies of oxygen and nutrients to cells within the engineered tissue construct. Soft-lithographic techniques have allowed the generation of hydrogel scaffolds containing a network of fluidic channels, but at the cost of complicated and often time-consuming manufacturing steps. We report a three-dimensional (3D) direct printing technique to construct hydrogel scaffolds containing fluidic channels. Cells can also be printed on to and embedded in the scaffold with this technique. Collagen hydrogel precursor was printed and subsequently crosslinked via nebulized sodium bicarbonate solution. A heated gelatin solution, which served as a sacrificial element for the fluidic channels, was printed between the collagen layers. The process was repeated layer-by-layer to form a 3D hydrogel block. The printed hydrogel block was heated to 37 degrees C, which allowed the gelatin to be selectively liquefied and drained, generating a hollow channel within the collagen scaffold. The dermal fibroblasts grown in a scaffold containing fluidic channels showed significantly elevated cell viability compared to the ones without any channels. The on-demand capability to print fluidic channel structures and cells in a 3D hydrogel scaffold offers flexibility in generating perfusable 3D artificial tissue composites.

The traditional notion that injured neurons are unable to regenerate in the adult mammalian brain and spinal cord has long been a concern. This view has led to methodology designed to overcome this problem, most recently by advancements in tissue engineering. Here, neural precursor cells (NPCs) and the Nogo receptor antibody (NgR-Ab) or poly-L-lysine (PLL) were tested in concert with hyaluronic acid hydrogel scaffolds (HA). In particular, we wished to optimize viability and differentiation of NPCs within HA hydrogel scaffolds. Our results show that HA hydrogels can be modified physically or chemically to improve NPCs attachment on the scaffolding doped with NgR-Ab or PLL. Both the HA hydrogels and their modifications support the viability of NPCs. NPCs were also able to differentiate into neurons and glial cells on HA hydrogels, although this was affected by the different modifications. Immunofluorescence showed that fewer β-III-tubulin antibody and antineurofilament antibody-positive cells were found on HA-PLL hydrogel compared with HA or HA NgR-Ab hydrogels. This indicates that the PLL-modified HA hydrogels may inhibit differentiation of NPCs, whereas modification by NgR-Ab had no such effect. Finally, the NgR-Ab-modified HA scaffold can be used as not only a NPC delivery system but also a bioactive factor transportation system for CNS repair. © 2009 Wiley-Liss, Inc.

Biological laser printing (BioLP) is a unique tool capable of printing high resolution two- and three-dimensional patterns of living mammalian cells, with greater than 95% viability. These results have been extended to primary cultured olfactory ensheathing cells (OECs), harvested from adult Sprague-Dawley rats. OECs have been found to provide stimulating environments for neurite outgrowth in spinal cord injury models. BioLP is unique in that small load volumes ( approximately microLs) are required to achieve printing, enabling low numbers of OECs to be harvested, concentrated and printed. BioLP was used to form several 8 mm lines of OECs throughout a multilayer hydrogel scaffold. The line width was as low as 20 microm, with most lines comprising aligned single cells. Fluorescent confocal microscopy was used to determine the functionality of the printed OECs, to monitor interactions between printed OECs, and to determine the extent of cell migration throughout the 3D scaffold. High-resolution printing of low cell count, harvested OECs is an important advancement for in vitro study of cell interactions and functionality. In addition, these cell-printed scaffolds may provide an alternative for spinal cord repair studies, as the single-cell patterns formed here are on relevant size scales for neurite outgrowth.

This work reports the preparation of silver nanoparticles (AgNPs) and synthesis of natural based hydrogel scaffold with an inclusion of AgNPs, chitosan/alginate/silver nanoparticles. The synthesised hydrogel scaffolds were characterised by using Fourier Transform Infrared Resonance Spectroscopy (FTIR). The FTIR result revealed that the shifting of the three peaks of 3252.95 cm −1 (–OH and –NH 2 stretching), 1591.33 cm −1 (C=O stretching) and 1411.88 cm −1 (N–H stretching) of chitosan/alginate/silver nanoparticles in compared to chitosan/alginate hydrogel indicating the presence of electrostatic interaction of –NH 3+ in chitosan reacted with the – COO– group of alginate and binding of the silver (Ag). These results indicated that chitosan/alginate/silver nanoparticles were consolidated in the composite system.

Understanding neural cell differentiation and neurite extension in three-dimensional scaffolds is critical for neural tissue engineering. This study explores the structure-function relationship between a 3D hydrogel scaffold and neural cell process extension and examines the role of ambient charge on neurite extension in 3D scaffolds. A range of agarose hydrogel concentrations was used to generate varied gel physical structures and the corresponding neurite extension was examined. Agarose gel concentration and the corresponding pore radius are important physical properties that influence neural cell function. The average pore radii of the gels were determined while the gel was in the hydrated state and in two different dehydrated states. As the gel concentration was increased, the average pore radius decreased exponentially. Similarly, the length of neurites extended by E9 chick DRGs cultured in agarose gels depends on gel concentration. The polycationic polysaccharide chitosan and the polyanionic polysaccharide alginate were used to incorporate charge into the 3D hydrogel scaffold, and neural cell response to charge was studied. Chitosan and alginate were covalently bound to the agarose hydrogel backbone using the bi-functional coupling agent 1,1'-carbonyldiimidazole. DRGs cultured in chitosan-coupled agarose gel exhibited a significant increase in neurite length compared to the unmodified agarose control. Conversely, the alginate-coupled agarose gels significantly inhibited neurite extension. This study demonstrates a strong, correlation between the ability of sensory ganglia to extend neurites in 3D gels and the hydrogel pore radius. In addition, our results demonstrate that charged biopolymers influence neurite extension in a polarity dependent manner.

A NEW TYPE OF PEPTIDIC hydrogel scaffold forms at polymer concentrations as low as 0.25 to 2.0% by weight, according to researchers at the Materials Research Laboratory at the University of California, Santa Barbara, and the department of materials science and engineering at the University of Delaware, Newark [Nature, 417, 424 (2002)]. The new hydrogels could be used in drug delivery and tissue engineering applications. The hydrogels are diblock copolypeptides with hydrophilic and hydrophobic regions. Usually, such a combination of hydrophilic and hydrophobic portions forms micelles, but not in this material. The hydrophobic polypeptide segments can be made to adopt different chain conformations, similar to those found in proteins, says team leader Timothy J. Deming of UCSB. "Ordered chain structures cannot pack efficiently into spheres" and so don't form micelles. Team members include, among others, graduate student Andrew P. Nowak at UCSB and Darrin Pochan, assistant professor at Delaware. One ...

Intervertebral disc (IVD) degeneration is one of the prevailing medical concern and actual health cost. Industries and academic institutions research for therapeutic solutions going from drugs to complete and partial arthroplasty devices as well as engineered tissues. Most research focus on the nucleus pulposus (NP) of the IVD because it is believed that, in the degeneration process, the NP is the scene of the first (irreversible) biochemical and mechanical changes. Mechanical, biochemical, nutritional and genetic factors may induce the degenerative process. Authors agree that (dynamic) mechanical stimuli are essential to preserve the integrity of the NP tissue. Indeed, sustained static loading and joint immobilization are proven to accelerate tissue degeneration. One possible explanation is that dynamic stimuli are required to transport (by convection) macromolecules, whereas smaller molecules are solely driven by passive diffusion. Another explanation is the requirement of direct cell-solid and/or cell-fluid interactions, namely mechanotransduction. In reality, both mechanisms are believed to influence tissue maintenance and should be considered evenly. It is, however, difficult to find a quantitative variable that unifies both theories. Viscoelasticity is the result of imperfect processes in which energy is dissipated. These imperfections arise from microstructural interactions and internal transport phenomena. Integrated, they result in time dependent and observable macroscopic behaviour. Although, interactions between phases are probably dominant, it makes sense to assume that cell-phase interactions are proportionally related. Therefore, investigating correlations between viscoelastic properties of the NP with cell synthesis may provide an experimental and theoretical mean to investigate the effect of dynamic mechanical stimuli on living tissues. The objective of this project is to verify this hypothesis on a hydrogel-cell construct model. Because the following step is to localize the viscoelastic description, extracellular matrix production is to be assessed with a 3D spatial resolution.