Engineering and cell attachment properties of human fibronectin-fibrinogen scaffolds for use in tissue engineered blood vessels.

Tissue engineered constructs reported to date have been prepared primarily from poly(glycolic) acid or collagen scaffolds onto which cells are grown and matured. In this paper we report experimental data to demonstrate the use of a natural, human protein, as a tubular scaffold for vascular grafting. Using a manual and a scalable dip-coating technique we prepared fibronectin-based tubes up to 12 cm in length and up to 3 mm in diameter. The tubes were flexible and their mechanical properties, measured in terms of tensile strength and burst pressure as a function of humidity, demonstrated their suitability as scaffolds for use in vascular grafting, e.g. coronary artery by pass grafting. In vitro tests involved the attachment of endothelial cells pumped under laminar flow conditions through the tube lumen and the adherence of smooth muscle cells on the outer surface of the tubes. These tests, carried out in multiwells, showed that the scaffolds had excellent cell attachment and guidance characteristics.

Wet extrusion of fibronectin-fibrinogen cables for application in tissue engineering.

A method for the wet extrusion of human plasma-derived fibronectin-fibrinogen cables is described. Solutions of fibronectin and fibrinogen with and without sodium alginate and carboxymethylcellulose (CMC) are tested. The rheological properties of the protein solutions changed from Newtonian to shear thinning non-Newtonian in the presence of small quantities of these additives, the apparent viscosity increased, and the extrusion properties of the protein solutions improved. Cables were prepared using a capillary with a diameter of 1 mm and overall length of 18 mm. Cable diameter was reduced to about 0.5 mm by drawing using a series of rollers. Cables prepared with sodium alginate were found to have suitable properties, and those made with CMC were sticky and difficult to handle. Solutions containing no sodium alginate required a minimum total protein concentration of about 70 mg/mL for extrusion. Extruded cables were prepared with solutions containing 140 mg/mL total protein with 12.9 mg/mL alginate (high protein), and 46 mg/mL total protein with 47.6 mg/mL of sodium alginate (high alginate). The mechanical strength of the extruded cables was within the range suitable for application in tissue engineering. Extrusion of the protein solutions into cables was achieved in a coagulation bath. Cables with a mechanical strength of approximately 30 N/mm(2), suitable for wound repair and nerve regeneration applications, were prepared with a coagulation bath containing 0.25 M HCl, 2% CaCl(2) at a pH of <0.9. These cables also had a large average elongation at break of 52%, and showed an increase in cable length after breakage (permanent set) of 20%, demonstrating the potential for drawing the cables down to a fine diameter.

The engineering effects of fluids flow on freely suspended biological macro-materials and macromolecules.

The manufacture of many biotechnologically important products requires consideration of the physical breakage and biochemical degradation pathways at all stages during processing, storage and transportation. The engineering flow environment in most items of bioprocess equipment has long been recognised as a key factor in determining these pathways and is the focus of the present review. Because of its industrial significance, the detrimental effects of the engineering flow environment on freely suspended bioparticles have been the subject of many scientific investigations over the past few decades. There is a general consensus of opinion that fluid shear and elongational stresses are the two main breakage pathways of relevance to processing of most biomaterials. An additional degradation pathway has also been identified involving significant losses of biological activity of macromolecules at gas-liquid, gas-solid and liquid-liquid interfaces. In such cases, the engineering flow field is shown to have a secondary role in determining the kinetics of inactivation. An equally important consideration in the optimisation of the relevant unit operations is the biomechanical integrity of the flow sensitive material. The biomechanical and biorheological parameters that determine the integrity of biomaterials are poorly defined, their evaluations present future research challenges and are of immediate engineering significance.

Quantitation of supercoiled circular content in plasmid DNA solutions using a fluorescence-based method.

A method for quantifying the proportion of supercoiled circular (SC) forms in DNA solutions is described. The method (SCFluo) takes advantage of the reversible denaturation property of SC forms and the high specificity of the PicoGreen fluorochrome for double-stranded (ds)DNA. Fluorescence values of forms capable of reversible denaturation after a 5 min heating, 2 min cooling step are normalised to fluorescence values of total dsDNA present in the preparation. For samples with a SC content >20-30%, good regression fits were obtained when values derived from densitometric scanning of an agarose gel and those derived from the SCFluo method were compared. The method represents an attractive alternative to currently established methods because it is simple, rapid and quantitative. During large-scale processing and long-term storage, enzymatic, chemical and shear degradation may substantially decrease the SC content of plasmid DNA preparations. Regulations for pharmaceutical grade products for use in gene therapy and DNA vaccination may require >90% of the plasmid to be in the SC form. In the present study the SC content of 6.9, 13 and 20 kb plasmid preparations that had been subjected to chemical and shear degradation was successfully quantified using the new method.

Removal of contaminant nucleic acids by nitrocellulose filtration during pharmaceutical-grade plasmid DNA processing.

Pharmaceutical-grade plasmid DNA for use in vaccines and gene therapy requires the development of reproducible and scaleable downstream processes. Shearing of chromosomal DNA at the commencement of the purification results in fragments that are difficult to separate from supercoiled plasmid DNA. Regulatory standards will probably require that the level of chromosomal DNA contamination is kept below 0.01 mg mg(-1) plasmid DNA. This work reports the use of nitrocellulose membranes to decrease chromosomal DNA contamination in plasmid DNA preparations derived from a 450-l bioreactor. Clarified lysates, resuspended PEG precipitates and anion exchange chromatography elutes were filtered through nitrocellulose. In all the cases, chromosomal DNA was selectively retained by the membrane while most supercoiled plasmid DNA was recovered in the filtrate. Contamination levels dropped from over 27% to below 1% as measured by Southern analysis. Under ionic strength conditions equal to or above 1.5 M NaCl, a fraction of the contaminant RNA was also retained by the nitrocellulose membrane.

Time course of SDS-alkaline lysis of recombinant bacterial cells for plasmid release.

SDS-alkaline lysis of recombinant Escherichia coli cell suspensions was carried out in a coaxial cylinder rheometer, and the data were used to establish the time course of lysis reaction. The results of the experiments showed that cell lysis reaction time depended on cell strain but was unaffected by plasmid size and plasmid copy number. The high molecular weight globular proteins and chromosomal DNA were denatured, and the resulting changes in rheometric measurements characterised the denaturation time.

High pressure disruption of yeast cells: The use of scale down operations for the prediction of protein release and cell debris size distribution.

Experiments were carried out aimed at establishing the effects of equipment scale down on the disruption of Baker's yeast cells in high pressure homogenisers. Data are reported on the cell debris particle size distribution (PSD) and on total protein release as a function of the applied pressure for two valve geometries and three scales of operation covering flow rates of 28, 60 and 280 L/h. A comparison of the results from the experiments indicates that over the range of parameters investigated both the total protein release and the cell debris PSDs are independent of valve geometry and flow rate through the homogeniser. These observations are discussed in the light of relevant previous publications. The cell debris PSDs have been simulated by using a recently published model and the total protein release data are described by the well-established Hetherington expression (Hetherington et al., 1971). (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 642-649, 1997.

Simulation of particle size distribution changes occurring during high-pressure disruption of bakers' yeast.

Measurements of size distributions are provided for the breakage of commercial packed bakers' yeast cells as a function of operating pressure and number of passes through a Manton Gaulin high-pressure homogenizer. A two parameter model was developed, based upon the use of a Boltzmann function, to simulate the changes in size distribution that accompany the cell breakage process. The effects of operating pressure and number of passes are incorporated in the model and the result is used to simulate the particle size distribution of the cell homogenate. The results show that there is little breakage below a threshold pressure of 115 bar and above which breakage is critically dependent upon the pressure and number of passes through the homogenizer. The analysis provides a means of studying the efficiency of centrifugation that may follow cell disruption and provides the basis for further studies of size distribution changes accompanying cell disruption.

Rheologies and morphologies of three actinomycetes in submerged culture.

The broth rheologies and morphologies of three actinomycetes (Saccharopolyspora erythraea, Actinomadura roseorufa, and Streptomyces rimosus) in submerged culture have been examined. The rheology of all the broths became pseudoplastic as soon as significant growth occurred with the power law index, n, falling to 0.20 to 0.25. The consistency index, K, rose with biomass concentration although in some instances it fell later in the fermentation. The mean main hyphal lengths of all cultures were in the range, 15 to 25 mum, and did not alter greatly even when large changes in K were occurring. (c) 1995 John Wiley & Sons, Inc.