Enhanced Neural Differentiation Using Simultaneous Application of 3D Scaffold Culture, Fluid Flow, and Electrical Stimulation in Bioreactors.

Neural differentiation is studied using a simultaneous application of 3D scaffold culture and hydrodynamic and electrical stimuli in purpose-designed recirculation bioreactors operated with continuous fluid flow. Pheochromocytoma (PC12) cells are seeded into nonwoven microfibrous viscose-rayon scaffolds functionalized with poly-l-lysine and laminin. Compared with the results from static control cultures with and without electrical stimulation and bioreactor cultures with the fluid flow without electrical stimulation, expression levels of the differentiation markers β3-tubulin, shootin1, and ephrin type-A receptor 2 are greatest when cells are cultured in bioreactors with fluid flow combined with in-situ electrical stimulus.

Interactivity of biochemical and physical stimuli during epigenetic conditioning and cardiomyocytic differentiation of stem and progenitor cells derived from adult hearts.

Mixed populations of cardiosphere-derived stem and progenitor cells containing proliferative and cardiomyogenically committed cells were obtained from adult rat hearts. The cells were cultured in either static 2D monolayers or dynamic 3D scaffold systems with fluid flow. Cardiomyocyte lineage commitment in terms of GATA4 and Nkx2.

Fast three-dimensional micropatterning of PC12 cells in rapidly crosslinked hydrogel scaffolds using ultrasonic standing waves.

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.

Electrical stimulation of cell growth and neurogenesis using conductive and nonconductive microfibrous scaffolds.

The effect of exogenous electrical stimulation on cell viability, attachment, growth, and neurogenesis was examined using PC12 cells in microfibrous viscose-rayon scaffolds immersed in culture medium. The scaffolds were applied either in their nonconductive state or after coating the fibres with 200 nm of gold to give a scaffold sheet resistivity of (13 ± 1.3) Ω square-1.

Production of zebrafish cardiospheres and cardiac progenitor cells in vitro and three-dimensional culture of adult zebrafish cardiac tissue in scaffolds.

The hearts of adult zebrafish (Danio rerio) are capable of complete regeneration in vivo even after major injury, making this species of particular interest for understanding the growth and differentiation processes required for cardiac tissue engineering. To date, little research has been carried out on in vitro culture of adult zebrafish cardiac cells. In this work, progenitor-rich cardiospheres suitable for cardiomyocyte differentiation and myocardial regeneration were produced from adult zebrafish hearts.

Biosynthesis of fluorescent CdS nanocrystals with semiconductor properties: Comparison of microbial and plant production systems.

This study investigated fission yeast (Schizosaccharomyces pombe) and hairy roots of tomato (Solanum lycopersicum) as in vitro production vehicles for biological synthesis of CdS quantum dots. Cd added during the mid-growth phase of the cultures was detoxified within the biomass into inorganic sulphide-containing complexes with the quantum confinement properties of semiconductor nanocrystals. Significant differences were found between the two host systems in terms of nanoparticle production kinetics, yield and quality.

Shear and Compression Bioreactor for Cartilage Synthesis.

Mechanical forces, including hydrodynamic shear, hydrostatic pressure, compression, tension, and friction, can have stimulatory effects on cartilage synthesis in tissue engineering systems. Bioreactors capable of exerting forces on cells and tissue constructs within a controlled culture environment are needed to provide appropriate mechanical stimuli. In this chapter, we describe the construction, assembly, and operation of a mechanobioreactor providing simultaneous dynamic shear and compressive loading on developing cartilage tissues to mimic the rolling and squeezing action of articular joints.

Mesenchymal Stem Cells Derived from Human Adipose Tissue.

Human adult mesenchymal stem cells are present in fat tissue, which can be obtained using surgical procedures such as liposuction. The multilineage capacity of mesenchymal stem cells makes them very valuable for cell-based medical therapies. In this chapter, we describe how to isolate mesenchymal stem cells from human adult fat tissue, propagate the cells in culture, and cryopreserve the cells for tissue engineering applications.

Human Fetal and Adult Chondrocytes.

As the only cell type found in healthy adult cartilage, chondrocytes are the obvious and most direct starting point for cartilage tissue engineering. Human adult, juvenile, neonatal, and fetal chondrocytes have all been demonstrated to produce cartilage matrix components in vitro for production of engineered tissues. In this chapter, procedures are outlined for isolation of chondrocytes from human fetal and adult cartilage.

Cartilage Tissue Engineering: What Have We Learned in Practice?

Many technologies that underpin tissue engineering as a research field were developed with the aim of producing functional human cartilage in vitro. Much of our practical experience with three-dimensional cultures, tissue bioreactors, scaffold materials, stem cells, and differentiation protocols was gained using cartilage as a model system. Despite these advances, however, generation of engineered cartilage matrix with the composition, structure, and mechanical properties of mature articular cartilage has not yet been achieved.

In situ generation of tunable porosity gradients in hydrogel-based scaffolds for microfluidic cell culture.

Compared with preformed anisotropic matrices, an anisotropic matrix that allows users to alter its properties and structure in situ after synthesis offers the important advantage of being able to mimic dynamic in vivo microenvironments, such as in tissues undergoing morphogenesis or in wounds undergoing tissue repair. In this study, porous gradients are generated in situ in a hydrogel comprising enzymatically crosslinked gelatin hydroxyphenylpropionic acid (GTN-HPA) conjugate and carboxylmethyl cellulose tyramine (CMC-TYR) conjugate. The GTN-HPA component acts as the backbone of the hydrogel, while CMC-TYR acts as a biocompatible sacrificial polymer.

Injectable 3D hydrogel scaffold with tailorable porosity post-implantation.

Since rates of tissue growth vary significantly between tissue types, and also between individuals due to differences in age, dietary intake, and lifestyle-related factors, engineering a scaffold system that is appropriate for personalized tissue engineering remains a significant challenge. In this study, a gelatin-hydroxyphenylpropionic acid/carboxylmethylcellulose-tyramine (Gtn-HPA/CMC-Tyr) porous hydrogel system that allows the pore structure of scaffolds to be altered in vivo after implantation is developed. Cross-linking of Gtn-HPA/CMC-Tyr hydrogels via horseradish peroxidase oxidative coupling is examined both in vitro and in vivo.

Metal uptake and nanoparticle synthesis in hairy root cultures.

: Hairy roots are a convenient experimental tool for investigating the interactions between plant cells and metal ions. Hairy roots of species capable of hyperaccumulating Cd and Ni have been applied to investigate heavy metal tolerance in plants; hairy roots of nonhyperaccumulator species have also been employed in metal uptake studies. Furnace treatment of hairy root biomass containing high concentrations of Ni has been used to generate Ni-rich bio-ore suitable for metal recovery in phytomining applications.

Therapeutically important proteins from in vitro plant tissue culture systems.

Plant cells cultured in liquid medium in bioreactors are now being used commercially to produce biopharmaceutical proteins. The emergence of in vitro plant cell culture as a production vehicle reflects the importance of key biosafety and biocontainment concerns affecting the competitiveness of alternative systems such as mammalian cell culture and agriculture. Food plant species are particularly attractive as hosts for in vitro protein production: the risk of transgene escape and food chain contamination is eliminated using containment facilities, while regulatory approval for oral delivery of drugs may be easier than if non-edible species were used.

Osteogenic differentiation and osteochondral tissue engineering using human adipose-derived stem cells.

Osteogenesis and the production of composite osteochondral tissues were investigated using human adult adipose-derived stem cells and polyglycolic acid (PGA) mesh scaffolds under dynamic culture conditions. For osteogenesis, cells were expanded with or without osteoinduction factors and cultured in control or osteogenic medium for 2 weeks. Osteogenic medium enhanced osteopontin and osteocalcin gene expression when applied after but not during cell expansion.

Three-dimensional nanocharacterization of porous hydrogel with ion and electron beams.

Porous hydrogels provide an excellent environment for cell growth and tissue regeneration, with high permeability for oxygen, nutrients, and other water-soluble metabolites through their high water-content matrix. The ability to image three-dimensional (3D) cell growth is crucial for understanding and studying various cellular activities in 3D context, particularly for designing new tissue engineering scaffold, but it is still challenging to study cell-biomaterial interfaces with high resolution imaging. We demonstrate using focused ion beam (FIB) milling, electron imaging, and associated microanalysis techniques that novel 3D characterizations can be performed effectively on cells growing inside 3D hydrogel scaffold.

The cartilage matrix molecule components produced by human foetal cartilage rudiment cells within scaffolds and the role of exogenous growth factors.

Development of cartilage lesions in osteoarthritis and following traumatic injury has important consequences on the weight bearing and articulation of joints, has severe impact on the quality of life of affected individuals and is of significant socioeconomic impact. Hyaline cartilage is a highly specialised tissue with a limited ability to self repair. Development of three-dimensional scaffolds which maintain the correct chondrocyte phenotype during expansion of cells in vitro and their application in regenerative strategies for cartilage repair is therefore a major research objective of many laboratories.

Tissue engineering of cartilage using a mechanobioreactor exerting simultaneous mechanical shear and compression to simulate the rolling action of articular joints.

The effect of dynamic mechanical shear and compression on the synthesis of human tissue-engineered cartilage was investigated using a mechanobioreactor capable of simulating the rolling action of articular joints in a mixed fluid environment. Human chondrocytes seeded into polyglycolic acid (PGA) mesh or PGA-alginate scaffolds were precultured in shaking T-flasks or recirculation perfusion bioreactors for 2.5 or 4 weeks prior to mechanical stimulation in the mechanobioreactor.

Chondrogenesis and cartilage tissue engineering: the longer road to technology development.

Joint injury and disease are painful and debilitating conditions affecting a substantial proportion of the population. The idea that damaged cartilage in articulating joints might be replaced seamlessly with tissue-engineered cartilage is of obvious commercial interest because the market for such treatments is large. Recently, a wealth of new information about the complex biology of chondrogenesis and cartilage has emerged from stem cell research, including increasing evidence of the role of physical stimuli in directing differentiation.

Strategies for enhancing the accumulation and retention of extracellular matrix in tissue-engineered cartilage cultured in bioreactors.

Production of tissue-engineered cartilage involves the synthesis and accumulation of key constituents such as glycosaminoglycan (GAG) and collagen type II to form insoluble extracellular matrix (ECM). During cartilage culture, macromolecular components are released from nascent tissues into the medium, representing a significant waste of biosynthetic resources. This work was aimed at developing strategies for improving ECM retention in cartilage constructs and thus the quality of engineered tissues produced in bioreactors.

Improved seeding of chondrocytes into polyglycolic acid scaffolds using semi-static and alginate loading methods.

Cell seeding and attachment in three-dimensional scaffolds is a key step in tissue engineering with implications for cell differentiation and tissue development. In this work, two new seeding methods were investigated using human chondrocytes and polyglycolic acid (PGA) fibrous mesh scaffolds. A simple semi-static seeding method using culture plates and tissue flasks was developed as an easy-to-perform modification of static seeding.

Extent of cell differentiation and capacity for cartilage synthesis in human adult adipose-derived stem cells: comparison with fetal chondrocytes.

This study evaluated the extent of differentiation and cartilage biosynthetic capacity of human adult adipose-derived stem cells relative to human fetal chondrocytes. Both types of cell were seeded into nonwoven-mesh polyglycolic acid (PGA) scaffolds and cultured under dynamic conditions with and without addition of TGF-beta1 and insulin. Gene expression for aggrecan and collagen type II was upregulated in the stem cells in the presence of growth factors, and key components of articular cartilage such as glycosaminoglycan (GAG) and collagen type II were synthesized in cultured tissue constructs.

Chondrogenic differentiation of human adipose-derived stem cells in polyglycolic acid mesh scaffolds under dynamic culture conditions.

Chondrogenic differentiation of human adult adipose-derived stem cells was studied in vitro for the development of engineered cartilage tissue. Cells cultured under dynamic conditions in polyglycolic acid (PGA) scaffolds produced substantially higher glycosaminoglycan (GAG) and total collagen levels than cells in pellet cultures. This result reflects the importance of cell attachment and cell-scaffold interactions in stem cell differentiation and chondrogenesis.