A New Strategy to Generate Functional Insulin-Producing Cell Lines by Somatic Gene Transfer into Pancreatic Progenitors

BACKGROUND: There is increasing interest in developing human cell lines to be used to better understand cell biology, but also for drug screening, toxicology analysis and future cell therapy. In the endocrine pancreatic field, functional human beta cell lines are extremely scarce. On the other hand, rodent insulin producing beta cells have been generated during the past years with great success. Many of such cell lines were produced by using transgenic mice expressing SV40T antigen under the control of the insulin promoter, an approach clearly inadequate in human. Our objective was to develop and validate in rodent an alternative transgenic-like approach, applicable to human tissue, by performing somatic gene transfer into pancreatic progenitors that will develop into beta cells. METHODS AND FINDINGS: In this study, rat embryonic pancreases were transduced with recombinant lentiviral vector expressing the SV40T antigen under the control of the insulin promoter. Transduced tissues were next transplanted under the kidney capsule of immuno-incompetent mice allowing insulinoma development from which beta cell lines were established. Gene expression profile, insulin content and glucose dependent secretion, normalization of glycemia upon transplantation into diabetic mice validated the approach to generate beta cell lines. CONCLUSIONS: Somatic gene transfer into pancreatic progenitors represents an alternative strategy to generate functional beta cell lines in rodent. Moreover, this approach can be generalized to derive cells lines from various tissues and most importantly from tissues of human origin

Biodegradation Studies of Novel Fluorinated Di-Vinyl Urethane Monomers and Interaction of Biological Elements with Their Polymerized Films

The monomeric components of resin composites in dental restorative materials are susceptible to hydrolysis in the oral cavity. The main objective of this study was to assess the bio-stability of fluorinated urethane dimethacrylates and determine the nature of fluoro-chemistry interactions with protein and bacterial adhesion (both sources of hydrolytic activity) onto cured resin. Degradation studies were performed in the presence of either albumin (in a mildly alkaline pH) or cholesterol esterase (CE). The surface chemistry of the polymers was assessed by water contact angle measurements, pre- and post- incubation with albumin. Adhesion of Streptococcus mutans to cured resin was investigated. The fluorinated monomers were more stable against degradation when compared to the commercial monomer bisphenol A-diglycidyl methacrylate (BisGMA). While fluorinated monomers showed hydrolytic stability with respect to CE, all fluorinated monomers underwent some degree of degradation with albumin. The fluoro-chemistry did not reduce protein and/or bacterial adhesion onto the surface, however post incubation with albumin, the fluorinated surfaces still presented hydrophobic character as determined by the high contact angle values ranging from 79° to 86°. These monomers could potentially be used to increase the hydrophobicity of polymeric composites and provide a means to moderate esterolytic degradation associated with the monomeric component of the polymers within the oral cavity

Alterations of MEK1/2-ERK1/2, IFNγ and Smad2/3 associated Signalling pathways during cryopreservation of ASCs affect their differentiation towards VSMC-like cells

Vascular smooth muscle cells (VSMCs) play essential roles in regulating blood vessel form and function and they are required for vascular tissue regeneration. Multipotent adipose derived stromal cells (ASCs) can be differentiated into VSMC-like cells, which can be used as a potential VSMC source for the development of vascular tissue. However, the effects of cryopreservation on the differentiation of ASCs towards VSMCs are poorly studied to date. This study compared fresh ASCs (FA) vs. cryopreserved ASCs (CA) with respect to their differentiation potential towards VSMC-like cells. The expression of contractile VSMC markers (such as smoothelin) and cell contractility were investigated. It was found that VSMC-like cells derived from CA expressed smoothelin gene and protein at lower levels and showed compromised contractility in response to vasoconstrictors, when compared with those derived from FA. Moreover, it was demonstrated that this negative effect of cryopreservation could be mediated by MEK1/2-ERK1/2, IFNγ and Smad2/3 associated Signalling pathways. Treatment of CA with MEK1/2-ERK1/2 activator or IFNγ neutralizing antibodies enhanced Smad2/3 phosphorylation and showed a rescue of the negative effect of cryopreservation on the differentiation of ASCs towards VSMC-like cells. These findings are important for defining approaches that may use cryopreserved ASCs for vascular tissue regeneration. Keywords: Adipose derived stromal cells, Vascular smooth muscle cell differentiation, Cryopreservation, Smoothelin, Interferon gamma, Extracellular signal regulated kinas

The Structure and Function of Next-Generation Gingival Graft Substitutes—A Perspective on Multilayer Electrospun Constructs with Consideration of Vascularization

There is a shortage of suitable tissue-engineered solutions for gingival recession, a soft tissue defect of the oral cavity. Autologous tissue grafts lead to an increase in morbidity due to complications at the donor site. Although material substitutes are available on the market, their development is early, and work to produce more functional material substitutes is underway. The latter materials along with newly conceived tissue-engineered substitutes must maintain volumetric form over time and have advantageous mechanical and biological characteristics facilitating the regeneration of functional gingival tissue. This review conveys a comprehensive and timely perspective to provide insight towards future work in the field, by linking the structure (specifically multilayered systems) and function of electrospun material-based approaches for gingival tissue engineering and regeneration. Electrospun material composites are reviewed alongside existing commercial material substitutes’, looking at current advantages and disadvantages. The importance of implementing physiologically relevant degradation profiles and mechanical properties into the design of material substitutes is presented and discussed. Further, given that the broader tissue engineering field has moved towards the use of pre-seeded scaffolds, a review of promising cell options, for generating tissue-engineered autologous gingival grafts from electrospun scaffolds is presented and their potential utility and limitations are discussed

Influence of biodegradable and non-biodegradable material surfaces on the differentiation of human monocyte-derived macrophages

Monocyte-derived macrophages (MDM) and multinucleated foreign body giant cells (FBGC) are the primary cell types that remain at the cell-material interface of polyurethane (PU)-based medical devices as a result of chronic inflammatory responses. In vitro studies have demonstrated that MDM possess degradative potential toward PU, which can result in device failure. Because most studies have followed the degradation potential, morphology, and function of these cells only once fully differentiated, the current study investigated the influence of a non-degradable control tissue culture-grade polystyrene (TCPS) surface relative to two degradable model polycarbonate-urethanes (PCNU), of different chemistry, on various parameters of MDM morphology and function during a 14-day differentiation time course. The differentiation of human monocytes isolated from whole blood on PCNU materials resulted in increased cell attachment, decreased multinucleation, and significant decreases in cell spreading when compared with cells differentiated on TCPS. Actin-stained podosome-like cell adhesion structures were increased in PCNU-adherent cells, accompanied by an alteration in β-actin and vinculin protein expression. The expression of the CD68 macrophage marker was reduced when cells were adherent to the PCNU materials and compared with TCPS, suggesting altered cell activation by the degradable relative to non-degradable materials. The degradative potential of these cells was altered by the material surface they were exposed to as measured by esterase activity and protein expression of monocyte-specific esterase. This was also supported by physical material breakdown evident in scanning electron microscopy images that illustrated holes in the PCNU films generated by the presence of differentiating MDM. It was concluded from these studies that PCNU materials significantly alter the function and morphology of differentiating MDM. This must be taken into consideration when studying cell-material interactions because these cells will receive cues from their immediate environment (including the biomaterial) upon differentiation, thereby affecting their resulting phenotype

Material surfaces affect the protein expression patterns of human macrophages: A proteomics approach

Monocyte-derived macrophages (MDM) are key inflammatory cells and are central to the foreign body response to implant materials. MDM have been shown to exhibit changes in actin cytoskeleton, multinucleation, cell size, and function in response to small alterations in polycarbonate-urethane (PCNU) surface chemistry. Although PCNU chemistry has an influence on de novo protein synthesis, no assessments of the protein expression profiles of MDM have yet been reported. The rapid emerging field of expression proteomics facilitates the study of changes in cellular protein profiles in response to their microenvironment. The current study applied proteomic techniques, 2-dimensional electrophoresis (2-DE) combined with MALDI-ToF (matrix assisted laser desorption ionization-time of flight) mass spectrometry, to determine differences in MDM protein expression influenced by PCNU. Results indicated that MDM responded to material chemistry by modulation of structural proteins (i.e. actin, vimentin, and tubulin). Additionally, intracellular protein modulation which requires proteins responsible for trafficking (i.e. chaperone proteins) and protein structure modification (i.e. bond rearrangement and protein folding) were also altered. This study demonstrated for the first time that a proteomics approach was able to detect protein expression profile changes in MDM cultured on different material surfaces, forming the basis for utilizing further quantitative proteomics techniques that could assist in elucidation of the mechanisms involved in MDM-material interaction. © 2006 Wiley Periodicals, Inc. J Biomed Mater Res, 200

<i>Streptococcus mutans</i> Proteases Degrade Dentinal Collagen

Here, we explored the role of S. mutans’s whole cell and discrete fractions in the degradation of type I collagen and dentinal collagen. Type I collagen gels and human demineralized dentin slabs (DS) were incubated in media alone or with one of the following: overnight (O/N) or newly inoculated (NEW) cultures of S. mutans UA159; intracellular proteins, supernatant or bacterial membranes of O/N cultures. Media from all groups were analyzed for protease-mediated release of the collagen-specific imino acid hydroxyproline. Images of type I collagen and DS were analyzed, respectively. Type I collagen degradation was highest for the supernatant (p p p S. mutans cultures (O/N and NEW), intracellular components, and supernatant. This study demonstrates that intracellular and extracellular proteolytic activities from S. mutans enable this cariogenic bacterium to degrade type I and dentinal collagen in a growth-phase dependent manner, potentially contributing to the progression of dental caries