Solution casting of chitosan membranes for in vitro evaluation of bioactivity.

To access publisher's full text version of this article, please click on the hyperlink in Additional Links field or click on the hyperlink at the top of the page marked Files. This article is open access.Considerable research is focusing on the surface modification of titanium implants for the treatment of orthopaedic tissue injuries to increase the success of orthopaedic fixations. Chitosan is one of the natural materials under investigation based on several favourable properties. Numerous techniques have been described for the preparation of chitosan membranes, including solution casting methods for the investigation of bioactivity before applying coatings onto potential titanium implants. Solution casting enables the easy in-house evaluation of chitosan membranes and allows for the selection of promising chitosan materials.We present a method for the standardized and easily applied preparation of chitosan membranes by solution casting. This protocol is suitable for chitosan materials spanning a wide degree of deacetylation, being derived from different chitin sources and chitosan derivatives with novel properties. We detail the preparation and quality control methods in order to prepare membranes with favourable bioactivity, sustaining cell attachment and proliferation for extended culture periods.The possibilities associated with the use of chitosan in tissue engineering applications are far from being exhausted and numerous challenges remain prior to successful translation into the clinics. Based on our experience, we have developed simple in-house methods for quality control of homogeneous membrane casting and early prediction of successful experimental outcome.Icelandic Research Fund 090007023 Icelandic Technology Development Fund 06136200

Chitosan and chitosan derivatives in tissue engineering and stem cell biology

Chitosan is a promising natural substances used in biomaterials research as it has several essential properties that can be applied in tissue engineering. This polymer can be easily combined with other biomaterials and it can be rapidly and economically processed to deliver growth factors and drugs. In the work presented in this thesis, the effect of natural, chitin-derived biomaterials on stem cell biology and osteogenic differentiation was determined and important properties of chitosan for tissue engineering applications were examined. Furthermore, it was evaluated how chitosan derivatives affect the expression and potentially regulate the chitinase-like protein YKL-40 in stem cells, which has been indicated to be involved in tissue remodeling, inflammation and disease pathogenesis. In paper I, we investigated the biological effects of the aminosugar glucosamine, which is the smallest, completely deacetylated subunit of chitin. Glucosamine is best known as a dietary supplement for chondro-protection, yet we were able to demonstrate that it upregulates the expression of osteogenic marker genes, which was strongly correlated to YKL-40 expression. This proposes a so far unknown role for YKL-40 in late-stage osteogenic differentiation. Chito-oligomers, derived from chitosan and chitin, are being increasingly studied owing to their bioactivity and water solubility. The biological potential is strongly dependent on the chemical properties and particularly hexamer and heptamer fractions are being considered most potent. The application of chito-oligomers is frequently limited to antitumor activity and inhibition of angiogenesis, but these chito-oligomers similarly affect gene expression and cytokine secretion, as described in paper II. The potency of hexamer fractions of chito-oligomers is strongly dependent on the degree of deacetylation, ultimately requiring the appropriate choice of chito-oligomer for any particular application. Endotoxin contamination is difficult to avoid during the handling of natural substances, and the biological effects of endotoxins on the body are extensive. Strict regulations are in place to reduce the risk of adverse health effects induced by medical devices, yet these recommendations remain inadequate and insufficiently specified. In paper III, we showed that endotoxin contamination in chitosan derivatives can result in false-positive results, completely altering product performance in vitro. In order to determine relevant properties of chitosan for tissue engineering applications, we prepared chitosan membranes as bioactive coatings. In paper IV, we compared chitosan membranes prepared from a wide range of degree of deacetylation and derived from different sources in terms of surface characteristics and bioactivity. This work resulted in paper V with the development of a standardized protocol for solution casting methods for chitosan membranes, in-house prediction of successful experimental outcome and long-term cell attachment comparable to commonly used tissue culture plastic

The Effect of Recombinant Human Interleukin-6 on Osteogenic Differentiation and YKL-40 Expression in Human, Bone Marrow-Derived Mesenchymal Stem Cells.

To access publisher's full text version of this article click on the hyperlink at the bottom of the pageHuman mesenchymal stem cells are an attractive cell source for tissue engineering and regenerative medicine applications, especially because of their differentiation potential toward the mesenchymal lineage. Furthermore, this cell type participates in the regeneration of tissue damage and plays an important role in immunity. Similarly, chitinase-like proteins have been proposed to aid in tissue remodeling, inflammation, and differentiation processes. The chitinase-like protein YKL-40 in particular is indicated in preventing damage to the extracellular matrix in response to proinflammatory cytokines, even though its biological function remains speculative. Finally, interleukin (IL)-6, a pleiotropic acute phase protein, participates in the regulation of bone turnover and immunoregulation. The physiological role of IL-6 in bone homeostasis is complex, exerting different effects on osteoblasts and osteoclasts depending on their differentiation stage. The aim of this study was to determine the effect of recombinant human IL-6 (5 ng/mL) on YKL-40 expression and osteogenic differentiation of human mesenchymal stem cells. Recombinant human IL-6 induced a donor-dependent change in mineralization and significantly promoted YKL-40 protein secretion. However, YKL-40 gene expression remained unaffected, and no statistically significant differences in the expression of osteogenic marker genes could be observed

November [picture] /

Also available in an electronic version via the Internet at: http://nla.gov.au/nla.pic-an14108969-32; Exhibited: "Shadows in the dust : a contemporary portrait of Aboriginal station life". Touring exhibition, 1996-1998.Peter Sellars, Willie Lawrence and their horses drink from the dam whilst the mob of cattle they've mustered find shade from the stifling November heat, Glen Garland Station, Queensland, 199

Paul Petroff as the Prince and Tamara Toumanova as the Queen of the Swans, in Le lac des cygnes, the Original Ballet Russe, Australian tour, His Majesty's Theatre, Melbourne, 1940 [picture] /

From: Le lac des cygnes (Swan lake) : choreographic poem in one act / music by Peter Ilich Tchaikovsky; Inscription: "3P/3".; Part of the collection: Hugh P. Hall collection of photographs, 1938-1940.; Performed March and April 1940.; Condition: Poor, silvering.; Choreography after M. Petipa ; scenery and costumes by C. Korovine ; scenery executed by O. Allegri.; Also available in an electronic version via the internet at: http://nla.gov.au/nla.pic-vn4175660. One of a collection of photographs taken by Hugh P. Hall of 28 ballet productions performed by the Covent Garden Russian Ballet (toured Australia 1938-1939) and the Original Ballet Russe (toured Australia 1939-1940). These are the second and third of the three Ballets Russes companies which toured Australasia between 1936 and 1940. The photographs were taken from the auditorium during a live performance in His Majesty's Theatre, Melbourne and mounted on cardboard for display purposes. For conservation and storage, the photographs have been demounted. The original arrangement of the photographs has been recorded, and details are available from the Pictures Branch of the National Library

Chitosan leads to downregulation of YKL-40 and inflammasome activation in human macrophages.

To access publisher's full text version of this article click on the hyperlink at the bottom of the pageChitosan, the deacetylated derivative of chitin, is used as biomaterial in diverse settings. It is also found on pathogens and can be proinflammatory. Shorter derivatives of chitosan can be generated chemically or enzymatically, chitosan oligosaccharides (ChOS). There is variation in the chemical composition of ChOS, including size distribution, but in general, they have been described as inert or anti-inflammatory. Active human chitinases can cleave chitin and chitosan, while inactive chitinases bind both but do not cleave. Both active and inactive chitinases have important roles in the immune response. The inactive chitinase YKL-40 is expressed highly during inflammation and has been proposed as a marker of poor prognosis. YKL-40 acts as a negative regulator of the inflammasome and as a positive regulator of angiogenesis. Levels of YKL-40 can therefore regulate levels of inflammation, the extent of angiogenesis, and the process of inflammation resolution. This study shows that chitosan leads to reduced secretion of YKL-40 by primary human macrophages and that this is concomitant with inflammasome activation. This was most pronounced with a highly deacetylated ChOS. No effect on the secretion of the active chitinase Chit-1 was detected. Smaller and more acetylated ChOS did not affect YKL-40 levels nor inflammasome activation. We conclude that this effect on the levels of YKL-40 is a part of the proinflammatory mechanisms of chitosan and its derivatives. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 103A: 2778-2785, 2015.Landspitali University Hospital Research Fund Icelandic Student Innovation Fund 132341009

Mesenchymal stem cell morphology during expansion and differentiation.

<p>MSC were stained with crystal violet after expansion in media containing 10% of FBS, HPLF or HPLO for three passages (A–C). MSC cultured in HPLF or HPLO developed more spindle shaped morphology than cells cultured in FBS, proliferated faster and left circular areas containing no cells between them. Differentiation towards osteoblasts, adipocytes and chondrocytes was initiated after expansion in the three supplements. After 28 days of osteogenic differentiation mineralization in the culture could be visualized with a Von Kossa staining (D–F). Successful adipogenic differentiation was confirmed with Oil red O staining (G–I) and after 28 days of chondrogenic pellet cultures, the pellets were sectioned and stained with toludine blue staining (J–L). Pictures representative of three experiments.</p

Reduction of mononuclear cell proliferation during co-culture with MSC.

<p>Mononuclear cells (MNC) were stimulated with phytohemaglutinin (PHA) and then cultured with or without MSC that had been previously expanded in media containing 10% of FBS, HPLF or HPLO. MNC proliferation was significantly less when co-cultured with MSC than if cultured on their own (p≤0.05, n = 3). The reduced proliferation was seen irrespective of the culture conditions the MSC had previously been exposed to. * = p≤0.05.</p

Expression of osteogenic marker genes during osteogenic differentiation of MSC.

<p>Osteogenic differentiation was initiated after MSC expansion in 10% of FBS, HPLF or HPLO. Expression of osteogenic marker genes was evaluated after 7 and 21 days of osteogenic differentiation and is represented with graphs for each gene (A–C, n = 3) and a heat-map (D) where green shades represent lower levels of expression and red shades higher levels of expression. <i>ALP</i> was evenly expressed throughout the differentiation (A,D) while the expression of <i>SPP1</i> increased from day 7 to day 21 (B,D), irrespective of the culture supplements the MSC had previously been exposed to (p≤0.01). Expression of <i>RUNX2</i> decreased from day 7 to day 21 for all cultures (C–D), still expression of <i>RUNX2</i> in cultures originating from HPLO treated MSC was higher at both time-points compared to FBS and also at day 21 for HPLF compared to FBS (p≤0.05). * = p≤0.05, ** = p≤0.01.</p