Stable production of hyaluronic acid in Streptococcus zooppidemicus chemostats operated at high dilution rate

Hyaluronic acid is routinely produced through fermentation of both Group A and C streptococci. Despite significant production costs associated with short fermentations and removal of contaminating proteins released during entry into stationary phase, hyaluronic acid is typically produced in batch rather than continuous culture. The main reason is that hyaluronic acid synthesis has been found to be unstable in continuous culture except at very low dilution rates. Here, we investigated the mechanisms underlying this instability and developed a stable, high dilution rate (0.4 h(-1)) chemostat process for both chemically defined and complex media operating for more than 150 h of production. In chemically defined medium, the product yield was 25% higher in chemostat cultures than in conventional batch culture when arginine or glucose was the limiting substrate. In contrast, glutamine limitation resulted in higher ATP requirements and a yield similar to that observed in batch culture. In complex, glucose-limited medium, ATP requirements were greatly reduced but biomass synthesis was favored over hyaluronic acid and no improvement in hyaluronic acid yield was observed. The successful establishment of continuous culture at high dilution rate enables both commercial production at reduced cost and a more rational characterization and optimization of hyaluronic acid production in streptococci. (c) 2005 Wiley Periodicals, Inc.

Microbial hyaluronic acid production

Hyaluronic acid (HA) is a commercially valuable medical biopolymer increasingly produced through microbial fermentation. Viscosity limits product yield and the focus of research and development has been on improving the key quality parameters, purity and molecular weight. Traditional strain and process optimisation has yielded significant improvements, but appears to have reached a limit. Metabolic engineering is providing new opportunities and HA produced in a heterologous host is about to enter the market. In order to realise the full potential of metabolic engineering, however, greater understanding of the mechanisms underlying chain termination is required.

A blank slate? Layer-by-layer deposition of hyaluronic acid and chitosan onto various surfaces

Although poly(alpha-hydroxy esters), especially the PLGA family of lactic acid/glycolic acid copolymers, have many properties which make them promising materials for tissue engineering, the inherent chemistry of surfaces made from these particular polymers is problematic. In vivo, they promote a strong foreign-body response as a result of nonspecific adsorption and denaturation of serum proteins, which generally results in the formation of a nonfunctional fibrous capsule. Surface modification post-production of the scaffolds is an often-utilized approach to solving this problem, conceptually allowing the formation of a scaffold with mechanical properties defined by the bulk material and molecular-level interactions defined by the modified surface properties. A promising concept is the so-called blank slate: essentially a surface that is rendered resistant to nonspecific protein adsorption but can be readily activated to covalently bind bio-functional molecules such as extracellular matrix proteins, growth factors or polysaccharides. This study focuses on the use of the quartz crystal microbalance (QCM) to follow the layer-by-layer (LbL) electrostatic deposition of high molecular weight hyaluronic acid and chitosan onto PLGA surfaces rendered positively charged by aminolysis, to form a robust, protein-resistant coating. We further show that this surface may be further functionalized via the covalent attachment of collagen IV, which may then be used as a template for the self-assembly of basement membrane components from dilute Matrigel. The response of NIH-3T3 fibroblasts to these surfaces was also followed and shown to closely parallel the results observed in the QCM.

Aerobic cultivation of Streptococcus zooepidemicus and the role of NADH oxidase

A metabolic flux model was developed for Streptococcus zooepidemicus to compare the metabolism of glucose and maltose during aerobic batch cultivation. Lactic acid was the main product of glucose metabolism whereas acetic acid was the main product of maltose metabolism. This difference was chiefly attributed to the two-fold higher flux through NADH oxidase in maltose-grown cells that enabled the ATP generation rate to remain high despite a slower maltose consumption rate. The two-fold higher flux was matched by a two-fold increase in NADH oxidase activity, 2.53 +/- 0.1 mumol NADH min(-1) mg(-1) protein on maltose versus 1.07 +/- 0.04 Rmol NADH min(-1) mg(-1) protein on glucose, indicating that NADH oxidase activity is regulated by the energy status of the cell. Surprisingly, the energy status of the cell had little impact on hyaluronic acid (HA) yield and molecular weight. (C) 2003 Elsevier Science B.V. All rights reserved.

Viscosupplementation for the treatment of osteoarthritis of the knee (Review)

Background Osteoarthritis (OA) is the most prevalent chronic joint disorder worldwide and is associated with significant pain and disability. Objectives To assess the effects of viscosupplementation in the treatment of OA of the knee. The products were hyaluronan and hylan derivatives (Adant, Arthrum H, Artz (Artzal, Supartz), BioHy (Arthrease, Euflexxa, Nuflexxa), Durolane, Fermathron, Go-On, Hyalgan, Hylan G-F 20 (Synvisc Hylan G-F 20), Hyruan, NRD-101 (Suvenyl), Orthovisc, Ostenil, Replasyn, SLM-10, Suplasyn, Synject and Zeel compositum). Search strategy MEDLINE (up to January (week 1) 2006 for update), EMBASE, PREMEDLINE, Current Contents up to July 2003, and the Cochrane Central Register of Controlled Trials (CENTRAL) were searched. Specialised journals and reference lists of identified randomised controlled trials (RCTs) and pertinent review articles up to December 2005 were handsearched. Selection criteria RCTs of viscosupplementation for the treatment of people with a diagnosis of OA of the knee were eligible. Single and double-blinded studies, placebo-based and comparative studies were eligible. At least one of the four OMERACT III core set outcome measures had to be reported (Bellamy 1997). Data collection and analysis Each trial was assessed independently by two reviewers for its methodological quality using a validated tool. All data were extracted by one reviewer and verified by a second reviewer. Continuous outcome measures were analysed as weighted mean differences (WMD) with 95% confidence intervals (CI). However, where different scales were used to measure the same outcome, standardized mean differences (SMD) were used. Dichotomous outcomes were analyzed by relative risk (RR). Main results Seventy-six trials with a median quality score of 3 (range 1 to 5) were identified. Follow-up periods varied between day of last injection and eighteen months. Forty trials included comparisons of hyaluronan/hylan and placebo (saline or arthrocentesis), ten trials included comparisons of intra-articular (IA) corticosteroids, six trials included comparisons of nonsteroidal anti-inflammatory drugs (NSAIDs), three trials included comparisons of physical therapy, two trials included comparisons of exercise, two trials included comparisons of arthroscopy, two trials included comparisons of conventional treatment, and fifteen trials included comparisons of other hyaluronans/hylan. The pooled analyses of the effects of viscosupplements against 'placebo' controls generally supported the efficacy of this class of intervention. In these same analyses, differential efficacy effects were observed for different products on different variables and at different timepoints. Of note is the 5 to 13 week post injection period which showed a percent improvement from baseline of 28 to 54% for pain and 9 to 32% for function. In general, comparable efficacy was noted against NSAIDs and longer-term benefits were noted in comparisons against IA corticosteroids. In general, few adverse events were reported in the hyaluronan/hylan trials included in these analyses. Authors' conclusions Based on the aforementioned analyses, viscosupplementation is an effective treatment for OA of the knee with beneficial effects: on pain, function and patient global assessment; and at different post injection periods but especially at the 5 to 13 week post injection period. It is of note that the magnitude of the clinical effect, as expressed by the WMD and standardised mean difference (SMD) from the RevMan 4.2 output, is different for different products, comparisons, timepoints, variables and trial designs. However, there are few randomised head-to-head comparisons of different viscosupplements and readers should be cautious, therefore, in drawing conclusions regarding the relative value of different products. The clinical effect for some products, against placebo, on some variables at some timepoints is in the moderate to large effect-size range. Readers should refer to relevant tables to review specific detail given the heterogeneity in effects across the product class and some discrepancies observed between the RevMan 4.2 analyses and the original publications. Overall, the analyses performed are positive for the HA class and particularly positive for some products with respect to certain variables and timepoints, such as pain on weight bearing at 5 to 13 weeks postinjection. In general, sample-size restrictions preclude any definitive comment on the safety of the HA class of products; however, within the constraints of the trial designs employed no major safety issues were detected. In some analyses viscosupplements were comparable in efficacy to systemic forms of active intervention, with more local reactions but fewer systemic adverse events. In other analyses HA products had more prolonged effects than IA corticosteroids. Overall, the aforementioned analyses support the use of the HA class of products in the treatment of knee OA.

Glucosamine supplementation during in vitro maturation inhibits subsequent embryo development: Possible role of the of developmental competence

Glucose concentration during cumulus-oocyte complex (COC) maturation influences several functions, including progression of oocyte meiosis, oocyte developmental competence, and cumulus mucification. Glucosamine (GlcN) is an alternative hexose substrate, specifically metabolized through the hexosamine biosynthesis pathway, which provides the intermediates for extracellular matrix formation during cumulus cell mucification. The aim of this study was to determine the influence of GlcN on meiotic progression and oocyte developmental competence following in vitro maturation (IVM). The presence of GlcN during bovine IVM did not affect the completion of nuclear maturation and early cleavage, but severely perturbed blastocyst development. This effect was subsequently shown to be dose-dependent and was also observed for porcine oocytes matured in vitro. Hexosamine biosynthesis upregulation using GlcN supplementation is well known to increase O-linked glycosylation of many intracellular signaling molecules, the best-characterized being the phosphoinositol-3-kinase (PI3K) signaling pathway. We observed extensive O-linked glycosylation in bovine cumulus cells, but not oocytes, following IVM in either the presence or the absence of GlcN. Inhibition of O-linked glycosylation significantly reversed the effect of GlcN-induced reduction in developmental competence, but inhibition of PI3K signaling had no effect. Our data are the first to link hexosamine biosynthesis, involved in cumulus cell mucification, to oocyte developmental competence during in vitro maturation.