Silk fibroin/gelatin microcarriers as scaffolds for bone tissue engineering.

Microcarrier cell scaffolds have potential as injectable cell delivery vehicles or as building blocks for tissue engineering. The use of small cell carriers allows for a 'bottom up' approach to tissue assembly when moulding microparticles into larger structures, which can facilitate the introduction of hierarchy by layering different matrices and cell types, while evenly distributing cells through the structure. In this work, silk fibroin (SF), purified from Bombyx mori cocoons, was blended with gelatin (G) to produce materials composed of varying ratios of the two components (SF: G 25:75, 50:50, and 75:25).

Zirconium amine tris(phenolate): A more effective initiator for biomedical lactide.

Here a zirconium amine tris(phenolate) is used as the initiator for the production of polylactide for biomedical applications, as a replacement for a tin initiator (usually tin octanoate). The ring opening polymerization (ROP) was carried out in the melt at 130°C. The zirconium-catalyzed PLA (PLA-Zr) required 30min, resulting in a polydispersity index (PDI) of 1.

An investigation into the stability of commercial versus MG63-derived hepatocyte growth factor under flow cultivation conditions.

The scale-up of tissue engineering cell culture must ensure that conditions are maintained while also being cost effective. Here we analyse the stability of hepatocyte growth factor (HGF) to investigate whether concentrations change under dynamic conditions, and compare commercial recombinant human HGF as an additive in 'standard medium', to HGF secreted by the osteosarcoma cell line MG63 as a 'preconditioned medium'. After 3 h under flow conditions, HGF in the standard medium degraded to 40% of its original concentration but HGF in the preconditioned medium remained at 100%.

Zscan4 is regulated by PI3-kinase and DNA-damaging agents and directly interacts with the transcriptional repressors LSD1 and CtBP2 in mouse embryonic stem cells.

The Zscan4 family of genes, encoding SCAN-domain and zinc finger-containing proteins, has been implicated in the control of early mammalian embryogenesis as well as the regulation of pluripotency and maintenance of genome integrity in mouse embryonic stem cells. However, many features of this enigmatic family of genes are poorly understood. Here we show that undifferentiated mouse embryonic stem cell (ESC) lines simultaneously express multiple members of the Zscan4 gene family, with Zscan4c, Zscan4f and Zscan4-ps2 consistently being the most abundant.

Perfusion bioreactor studies of chondrocyte growth in alginate-chitosan capsules.

The combination of progenitor cells, cell-friendly scaffold, and a three-dimensional culture system has been investigated for the culture of cartilage tissue. We have assessed chondrogenesis of alginate-chitosan-encapsulated STRO-1-isolated human mesenchymal progenitor cells. In addition, ATDC-5 cells and human articular chondrocytes were also evaluated.

The effect of porosity of a biphasic ceramic scaffold on human skeletal stem cell growth and differentiation in vivo.

Skeletal stem cell (SSC) growth on a novel porous HA/TCP scaffold has been investigated in vivo. The effect of porosity on osteogenic differentiation was assessed by comparing two groups of scaffolds with differing porosity but controlled pore size. Histology, microCT, scanning electron microscopy, and biochemical analysis were used to assess SSC proliferation and differentiation.

A theoretical approach to zonation in a bioartificial liver.

Bioartificial livers have yet to gain clinical acceptance. In a previous study, a theoretical model was utilized to create operating region charts that graphically illustrated viable bioartificial liver configurations. On this basis a rationale for the choice of operating and design parameters for the device was created.

Surfactant-free poly(lactide-co-glycolide) honeycomb films for tissue engineering: relating solvent, monomer ratio and humidity to scaffold structure.

One-step surfactant-free, water-droplet templating has been developed as a fabrication method for a poly(lactide-co-glycolide) (PLGA) film that can be used as a model to investigate the relationship between solvent, monomer ratio, polymer concentration and humidity on its structure. The resulting material is a honeycomb-structured film. Formation of this structure was highly sensitive to solvent, monomer ratio, polymer concentration and humidity.

Three-dimensional culture systems for the expansion of pluripotent embryonic stem cells.

Mouse embryonic stem cell (ESC) lines, and more recently human ESC lines, have become valuable tools for studying early mammalian development. Increasing interest in ESCs and their differentiated progeny in drug discovery and as potential therapeutic agents has highlighted the fact that current two-dimensional (2D) static culturing techniques are inadequate for large-scale production. The culture of mammalian cells in three-dimensional (3D) agitated systems has been shown to overcome many of the restrictions of 2D and is therefore likely to be effective for ESC proliferation.

A theoretical method to improve and optimize the design of bioartificial livers.

Bioartificial livers (BALs) are a potentially effective countermeasure against liver failure, particularly in cases of acute or fulminant liver failure. It is hoped these devices can sustain a patient's liver function until recovery or transplant. However, no large-scale clinical trial has yet proven that BALs are particularly effective and evidently design issues remain to be addressed.

Post-culture treatment protocols for PLGA membrane scaffolds.

The interactions of post-culture treatments reagents used for fixing, lysing and cell quantification on poly(lactide-co-glycolide) (PLGA) flat sheet membrane scaffolds are presented. Lysing with Alkaline buffer solution/Triton X-100/MilliQ water (ATM) and fixing with 10% Neutral Buffered Formalin (10% NBF) had no affect on membrane structure while fixing with 95% ethanol caused smoothing of the surface, shrinkage and a reduction in surface area of 55, 48 and 33, for 100:0, 75:25 and 50:50 (PLA:PGA), respectively. PicoGreen assay was selected for cell (560pZIPv.

An in vitro study of electrically active hydroxyapatite-barium titanate ceramics using Saos-2 cells.

Electrically active ceramics are of interest as bone graft substitute materials. This study investigated the ferroelectric properties of hydroxyapatite-barium titanate (HABT) composites and the behaviour of osteoblast-like cells seeded on their surfaces. A piezoelectric coefficient (d(33)) of 57.

Validity of DNA analysis to determine cell numbers in tissue engineering scaffolds.

The potential of a DNA content assay, PicoGreen, for use in 3D bioengineered constructs was examined. The assay was tested on ATDC5 cells in situ during culture in typical tissue engineering 3D constructs. Comparisons of cell standards from cell lines and primary cells to lambdaDNA standards was also conducted.

Formation of a human-derived fat tissue layer in P(DL)LGA hollow fibre scaffolds for adipocyte tissue engineering.

Development of adipose tissue-engineering strategies, where human bone marrow stromal cells (HBMSC) are combined with three-dimensional scaffolds, is likely to prove valuable for soft tissue restoration. In this study, we assessed the function of poly(DL-lactide-co-glycolide) (P(DL)LGA) hollow fibres in facilitating the development of HBMSC-derived adipocytes for advancement of an associated adipocyte layer. The large surface area of 75:25 P(DL)LGA fibres facilitated the rapid generation of extensive adipocyte aggregates from an undifferentiated HBMSC monolayer, where the fat-laden cells stained positive with Oil Red O and expressed the adipocyte marker, fatty acid binding protein 3 (FABP3).

Mathematical modelling of fibre-enhanced perfusion inside a tissue-engineering bioreactor.

We develop a simple mathematical model for forced flow of culture medium through a porous scaffold in a tissue-engineering bioreactor. Porous-walled hollow fibres penetrate the scaffold and act as additional sources of culture medium. The model, based on Darcy's law, is used to examine the nutrient and shear-stress distributions throughout the scaffold.

MagicWand: a single, designed peptide that assembles to stable, ordered alpha-helical fibers.

We describe a straightforward single-peptide design that self-assembles into extended and thickened nano-to-mesoscale fibers of remarkable stability and order. The basic chassis of the design is the well-understood dimeric alpha-helical coiled-coil motif. As such, the peptide has a heptad sequence repeat, abcdefg , with isoleucine and leucine residues at the a and d sites to ensure dimerization.

Human bone derived cell culture on PLGA flat sheet membranes of different lactide:glycolide ratio.

Providing a scaffold that can supply nutrients on a large scale (several cubic centimeters) is the key to successfully regenerating vascularized tissue: biodegradable membranes are a promising new scaffold suited to this purpose. Poly(lactic-co-glycolic-acid) (PLGA) flat sheet membranes of different lactide:glycolide ratios, prepared by phase inversion using 1-methyl-2-pyrrolidinone (NMP) as the solvent and water as the nonsolvent, were compared by assessing attachment, proliferation and osteogenic function of human bone derived cells (HBDC). Three different lactide:glycolide ratios, 50:50, 75:25, and 100:0, were compared to tissue culture polystyrene (TCPS).

Vascular tissue engineering: bioreactor design considerations for extended culture of primary human vascular smooth muscle cells.

The influence of mechanical stimulation on cell populations not only helps maintain the specific cellular phenotype but also plays a significant role during differentiation and maturation of plastic cells. This is particularly true of tissue-engineered vascular tissue, where in vivo shear forces at the blood interface help maintain the function of the endothelium. Considerable effort has gone into the design and implementation of functional bioreactors that mimic the chemical and mechanical forces associated with the in vivo environment.

Expansion of human bone marrow stromal cells on poly-(DL-lactide-co-glycolide) (PDL LGA) hollow fibres designed for use in skeletal tissue engineering.

Strategies to expand human bone marrow stromal cells (HBMSC) for bone tissue engineering are a key to revolutionising the processes involved in three-dimensional skeletal tissue reconstruction. To facilitate this process we believe the use of biodegradable porous poly(DL-lactide-co-glycolide) (PDL LGA) hollow fibres as a scaffold used in combination with HBMSC to initiate natural bone repair and regeneration offers a potential solution. In this study, the biocompatibility of 75:25 PDL LGA fibres with HBMSC and the capacity of a PDL LGA fibre-associated HBMSC-monolayer to establish an osteogenic phenotype in vivo was examined.

Strategies to promote chondrogenesis and osteogenesis from human bone marrow cells and articular chondrocytes encapsulated in polysaccharide templates.

The aim of this study was to synthesize functional in vitro and in vivo 3-dimensional (3D) constructs using a mix of human mesenchymal populations and articular chondrocytes encapsulated in biomineralized polysaccharide templates. Single-cell-type populations or mixtures of both cell types were encapsulated in alginate/chitosan and cultured within a rotating-bioreactor, perfused bioreactor system, or static conditions for 28 days. Within single cell-type populations, type II collagen immunopositive cells were present within lacunae in rotating-bioreactor capsules, with an increased proportion of metabolically active cells compared with perfused and static constructs.

Effects of common sterilization methods on the structure and properties of poly(D,L lactic-co-glycolic acid) scaffolds.

While methods for the production of scaffolds with the appropriate mechanical properties and architecture for tissue engineering are attracting much attention, the effects of subsequent sterilization processes on the scaffold properties have often been overlooked. This study sought to determine the effects of sterilization with ethanol, peracetic acid, ultraviolet irradiation, and antibiotic solution on the structure of 50:50 (mol:mol) 65:35, and 85:15 poly(D,L-lactic-co-glycolic acid [PLGA]) flat-sheet and hollow-fiber scaffolds. All methods resulted in scaffold sterilization, but scanning electron microscopy revealed deformations to the scaffold surface for all treatments.

Poly(lactic-co-glycolic acid) hollow fibre membranes for use as a tissue engineering scaffold.

Mass transfer limitations of scaffolds are currently hindering the development of 3-dimensional, clinically viable, tissue engineered constructs. We have developed a poly(lactide-co-glycolide) (PLGA) hollow fibre membrane scaffold that will provide support for cell culture, allow psuedovascularisation in vitro and provide channels for angiogenesis in vivo. We produced P(DL)LGA flat sheet membranes using 1, 4-dioxane and 1-methyl-2-pyrrolidinone (NMP) as solvents and water as the nonsolvent, and hollow fibre membranes, using NMP and water, by dry/wet- and wet-spinning.

A comparison of colorimetric and DNA quantification assays for the assessment of meniscal fibrochondrocyte proliferation in microcarrier culture.

We have developed and refined a rapid, reliable method for the evaluation of attachment and proliferation of ovine meniscal chondrocytes in microcarrier culture. Assays measuring both mitochondrial activity, using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] and MTS [3-(4,5-dimethylthiazol-2-yl)5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium], and DNA synthesis with a PicoGreen assay were compared. The MTT assay was the most sensitive at lower cell concentrations and enabled accurate assessment of cell proliferation over 14 day culture.

Endothelial and smooth muscle cell seeding onto processed ex vivo arterial scaffolds using 3D vascular bioreactors.

Biomaterials derived from ex vivo tissues offer a viable alternative to synthetic materials for organ replacement therapies. In this study, we describe the use of a tissue engineering scaffold derived from ex vivo arterial tissue to assess vascular cell adhesion within a three-dimensional perfusion bioreactor. With the aim of maximizing seeding efficiency, five methods for endothelial cell (EC) and three independent methods for vascular smooth muscle cell (VSMC) adhesion were explored.

Porcine collagen crosslinking, degradation and its capability for fibroblast adhesion and proliferation.

Porcine dermal collagen permanently crosslinked with hexamethylene diisocyanate was investigated for its suitability as a dermal tissue engineering matrix. It was found that the chemically crosslinked collagen had far fewer free lysine groups per collagen molecule than did the uncrosslinked matrix. The ability of the matrix to support human primary fibroblast outgrowth from explants was compared for matrices that had been presoaked in various solutions, including fibroblast media, cysteine and phosphate buffered saline (PBS).