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). Cell compatibility to these materials was first confirmed in two-dimensional culture and found to be equivalent to standard tissue culture plastic, and better than SF or G alone. The mechanical properties of the blends were investigated and the blended materials were found to have increased Young's moduli over SF alone. Microcarriers of SF/G blends with defined diameters were generated in a reproducible manner through the use of an axisymmetric flow focussing device, constructed from off-the-shelf parts and fittings. These SF/G microcarriers supported adhesion of rat mesenchymal stem cells with high degrees of efficiency under dynamic culture conditions and, after culturing in osteogenic differentiation medium, cells were shown to have characteristics typical of osteoblasts. This work illustrates that microcarriers composed of SF/G blends are promising building blocks for osteogenic tissue engineering.

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 1.17, compared to 1h and PDI=1.77 for tin-catalyzed PLA (PLA-Sn). PLA-Zr and PLA-Sn supported osteosarcoma cell (MG63) culture to the same extent (cell number, morphology, extracellular matrix production and osteogenic function) until day 14 when the PLA-Zr showed increased cell number, overall extracellular matrix production and osteogenic function. To conclude, the reduction in reaction time, controllable microstructure and biologically benign nature of the zirconium amine tris(phenolate) initiator shows that it is a more effective initiator for ROP of polylactide for biomedical applications.

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%. The concentration of secreted HGF was 10 times greater than the working concentration of commercially-available HGF. Thus HGF within this medium has increased stability; MG63-derived HGF should therefore be investigated as a cost-effective alternative to current lyophilised powders for use in in vitro models. Furthermore, we recommend that those intending to use HGF (or other growth factors) should consider similar stability testing before embarking on experiments with media flow.

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. Despite this, between only 0.1 and 0.7% of undifferentiated mouse pluripotent stem cells express Zscan4 protein at a given time, consistent with a very restricted pattern of Zscan4 transcripts reported previously. Herein we demonstrate that Zscan4 expression is regulated by the p110α catalytic isoform of phosphoinositide 3-kinases and is induced following exposure to a sub-class of DNA-damage-inducing agents, including Zeocin and Cisplatin. Furthermore, we observe that Zscan4 protein expression peaks during the G2 phase of the cell cycle, suggesting that it may play a critical role at this checkpoint. Studies with GAL4-fusion proteins suggest a role for Zscan4 in transcriptional regulation, further supported by the fact that protein interaction analyses demonstrate that Zscan4 interacts with both LSD1 and CtBP2 in ESC nuclei. This study advances and extends our understanding of Zscan4 expression, regulation and mechanism of action. Based on our data we propose that Zscan4 may regulate gene transcription in mouse ES cells through interaction with LSD1 and CtBP2.

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. The 45 pores per inch (ppi) scaffold demonstrated a greater increase in density than the 30 ppi scaffold following in vivo culture, and a reduction in dimensions of the pores and channels of the higher porosity scaffold was observed, indicating generation of new tissue within the pores. All scaffolds supported SSC proliferation but the higher scaffold porosity augmented osteogenic differentiation. ALP specific activity was enhanced on the 45 ppi scaffold compared to the 30 ppi scaffold. These studies demonstrate the importance of porosity in scaffold design and impact therein for tissue engineering application.

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. We have used a novel 3D bioreactor system that enabled perfusion of the capsules with culture medium up to 28 days. Results from culturing all cell types indicated chondrogenesis, both in static and bioreactor culture. The expression of SOX-9 and type II collagen was examined as a marker of differentiation. ATDC-5s expressed both SOX-9 and type II collagen under perfused and static culture conditions. In monolayer cell culture, human articular chondrocytes did not express either SOX-9 or type II collagen and STRO-1 expressed alkaline phosphatase, indicating osteogenesis. However, when these cells were encapsulated in alginate-chitosan, both expressed SOX-9 under static and perfused cultures, but type II collagen was only expressed under perfused culture conditions. We have also noted that the perfusion rates used were too low to ensure a significant advantage over static culture, but that use of the bioreactor eliminated the need for manual feeding and intervention of the cells over the 28-day period.

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. The concept is extended here to include aspects of liver zonation for further design optimization. In vivo, liver cells display heterogeneity with respect to metabolic activity according to their position in the liver lobule. It is thought that oxygen tension is a primary modulator of this heterogeneity and on this assumption a theoretical model to describe the metabolic zonation within an in vitro bioartificial liver device has been adopted. The distribution of the metabolic zones under varying design and operating parameters is examined. In addition, plasma flow rates are calculated that give rise to an equal distribution of the metabolic zones. The results show that when a clinically relevant number of cells are contained in the BAL (10 billion), it is possible to constrain each of the three metabolic zones to approximately one-third of the cell volume. This is the case for a number of different bioreactor designs. These considerations allow bioartificial liver design to be optimized.

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. Surfactant-free, water-droplet templating thus allows investigation of fabrication parameters and that PLGA monomer ratio selection is important for scaffold structure but not for MG63 cell attachment and proliferation.

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. Using murine ESCs as our initial model, we investigated the effectiveness of different 3D culture environments for the expansion of pluripotent ESCs. Solohill Collagen, Solohill FACT, and Cultispher-S microcarriers were employed and used in conjunction with stirred bioreactors. Initial seeding parameters, including cell number and agitation conditions, were found to be critical in promoting attachment to microcarriers and minimizing the size of aggregates formed. While all microcarriers supported the growth of undifferentiated mESCs, Cultispher-S out-performed the Solohill microcarriers. When cultured for successive passages on Cultispher-S microcarriers, mESCs maintained their pluripotency, demonstrated by self-renewal, expression of pluripotency markers and the ability to undergo multi-lineage differentiation. When these optimized conditions were applied to unweaned human ESCs, Cultispher-S microcarriers supported the growth of hESCs that retained expression of pluripotency markers including SSEA4, Tra-1-60, NANOG, and OCT-4. Our study highlights the importance of optimization of initial seeding parameters and provides proof-of-concept data demonstrating the utility of microcarriers and bioreactors for the expansion of hESCs.

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. One aspect of BAL design that must be considered is the mass transfer of adequate oxygen to the hepatocytes within the device. We present here a mathematical modeling approach to oxygen mass transport in a BAL. A mathematical model based upon Krogh cylinders is outlined to describe a diffusion-limited hollow fiber bioreactor. In addition, operating constraints are defined on the system--cells should not experience hypoxia and the cell population should be of adequate size. By combining modeling results with these operating constraints and presenting the results graphically, "operating region" charts can be constructed for the hollow fiber BAL (HF-BAL). The effects of varying various operating parameters on the BAL are then established. It is found that smaller radii and short, thin walled fibers are generally advantageous while cell populations in excess of 10 billion could be supported in the BAL with a plasma flow rate of 200 mL/min. For fibers of intermediate length and lumen radius, the minimum number of fibers required to produce a viable design ranges approximately from 7,000-10,000. In theory, this may be enough to support patients with failing livers.

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.neo) quantification since the background noise would not affect readings for cell numbers over 3,000 cells/cm(2), while the background reading was too high for MTT and Methylene Blue (MB). MB at 0.5% (w/v) was, however, deemed suitable for visualising cell morphology on the membranes. Furthermore ATM buffer was suitable for the PicoGreen assay, which allows the same samples to be used for quantification of alkaline phosphatase activity.

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 57.8 pCN(-1) was observed in HABT discs prepared for cell culture. The attachment, proliferation, viability, morphology and metabolic activity of cells cultured on unpoled HABT were comparable to those observed on commercially available hydroxyapatite at all time points. No indication of the cytotoxicity of HABT was detected. At one day after seeding, cell attachment was modified on both the positive and negative surfaces of poled HABT. After longer incubations, all parameters observed were comparable on poled and unpoled ceramics. The results indicate that HABT ceramics are biocompatible in the short term in vitro and that further investigation of cell responses to these materials under mechanical load and at longer incubation times is warranted.

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. An effective working range of the assay within 3D constructs was shown up to 2.5 x 10(5) cells ml(-1). From significant variation found in DNA content between cell lines and primary cells, it was concluded that the most accurate standard to use for the assay was from the cell type being examined.

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). Following implantation subcutaneously in severely compromised immunodeficient mice, the adipogenic phenotype of the PLGA-adipocyte graft was maintained for up to 56 days. Confocal microscopy showed associated LipidTOX Deep Red neutral lipid staining in an (FL)P(DL)LGA fibre-adipocyte graft after 56 days, critical evidence demonstrating maintenance of the adipocyte phenotype in the subcutaneous graft. To support adipose tissue advancement in a defined volume, the P(DL)LGA-adipocyte scaffold was encapsulated within alginate/chitosan hydrogel capsules (typical diameters, 4.0 mm). In a 28-day in vivo trial in immunodeficient mice, clusters of the capsules were maintained at the subcutaneous site. An adipocyte tissue layer advancing within the surrounding hydrogel was demonstrated.

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. We consider several configurations of fibres and inlet and outlet pipes. Compared with a numerical solution of the full Navier-Stokes equations within the complex scaffold geometry, the modelling approach is cheap, and does not require knowledge of the detailed microstructure of the particular scaffold being used. The potential of this approach is demonstrated through quantification of the effect the additional flow from the fibres has on the nutrient and shear-stress distribution.

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. In addition, to direct staggered assembly of peptides and to foster fibrillogenesisthat is, as opposed to blunt-ended discrete speciesthe terminal quarters of the peptide are cationic and the central half anionic with lysine and glutamate, respectively, at core-flanking e and g positions. This +,-,-,+ arrangement gives the peptide its name, MagicWand (MW). As judged by circular dichroism (CD) spectra, MW assembles to alpha-helical structures in the sub-micromolar range and above. The thermal unfolding of MW is reversible with a melting temperature >70 degrees C at 100 muM peptide concentration. Negative-stain transmission electron microscopy (TEM) of MW assemblies reveals stiff, straight, fibrous rods that extended for tens of microns. Moreover, different stains highlight considerable order both perpendicular and parallel to the fiber long axis. The dimensions of these features are consistent with bundles of long, straight coiled alpha-helical coiled coils with their axes aligned parallel to the long axis of the fibers. The fiber thickening indicates inter-coiled-coil interactions. Mutagenesis of the outer surface of the peptide i.e., at the b and f positionscombined with stability and microscopy measurements, highlights the role of electrostatic and cation-pi interactions in driving fiber formation, stability and thickening. These findings are discussed in the context of the growing number of self-assembling peptide-based fibrous systems.

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). For attachment, 50:50 and 75:25 had similar numbers to TCPS but 100:0 had significantly fewer cells than TCPS. 50:50 and 75:25 had significantly lower HBDC numbers after 7 days but 100:0 had similar numbers compared to TCPS. For proliferation the cell number on the membranes were similar to each other. After 3 weeks, osteoblastic function of the HBDC, shown by mineralization and alkaline phosphatase activity, was present but was significantly lower compared to the TCPS control but similar when the membranes were compared. PLGA membranes fabricated from a range of ratios support HBDC culture so the optimum scaffold composition can be selected based on other factors, such as degradation rate.

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. Using a decellularized ex vivo porcine carotid artery as a model scaffold, we describe a number of important design criteria used to develop a vascular perfusion bioreactor and its supporting process-flow. The results of a comparative analysis of primary human vascular smooth muscle cells cultured under traditional"static conditions" and "dynamic loading" are described, where the expression of MMP-2 and 9 and cathepsin-L were assessed. Continued design improvements to perfusion bioreactors may improve cellular interactions, leading to constructs with improved biological function.

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. A high proportion of HBMSC survived when expanded on PDL LGA fibres for 6 days, with only 10% of the propidium iodide (pI)-labelled population represented in the sub-G1 DNA peak on analysis by flow cytometry. Tracking carboxy-fluorescein diacetate, succinimidyl ester (CFSE)-labelled HBMSC by flow cytometry indicated that HBMSC attachment to the P(DL)LGA fibres does not interfere with their rate of proliferation. Furthermore, in response to osteogenic stimuli, HBMSC expanded on PDL LGA fibres can differentiate, as expected, along the osteogenic lineage with associated alkaline phosphatase activity. Following implantation into SCID mice, osteogenic-conditioned PDL LGA fibre-HBMSC graft resulted in type I collagen deposition and associated bone mineralisation and osteoid formation, as evidenced by immunohistochemistry and histology. These studies provide evidence that porous PDL LGA hollow fibre-HBMSC graft is an innovative biomaterial that offers new approaches to mesenchymal cell expansion, which could be utilised as a scaffold for skeletal tissue generation.

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. The resulting fibres had an outer diameter of 700 micro m and an inner diameter of 250 micro m with 0.2-1.0 micro m pores on the culture surface. It was shown that varying the air gap and temperature when spinning changed the morphology of the fibres. The introduction of a 50 mm air gap caused a dense skin of 5 micro m thick to form, compared to a skin of 0.5 micro m thick without an air gap. Spinning at 40 degrees C produced fibres with a more open central section in the wall that contained more, larger macrovoids compared to fibres spun at 20 degrees C. Culture of the immortalised osteogenic cell line 560pZIPv.neo (pZIP) was carried out on the P(DL)LGA flat sheets in static culture and in a P(DL)LGA hollow fibre bioreactor under counter-current flow conditions. Attachment and proliferation was statistically similar to tissue culture polystyrene on the flat sheets and was also successful in the hollow fibre bioreactor. The P(DL)LGA hollow fibres are a promising scaffold to address the size limitations currently seen in tissue engineered constructs.