Cross talk between the extracellular matrix and the immune system in the context of endocrine pancreatic islet transplantation. A review article.

This review aims to highlight the importance of the bidirectional influence of the extracellular matrix (ECM) and immune cells in the context of type 1 diabetes mellitus (T1DM) and endocrine pancreatic islet transplantation. We introduced the main classes of molecules and proteins constituting the ECM as well as cells and cytokines of the immune system with the aim to further examine their roles in T1DM and islet transplantation. Integrins expressed by immune cells and their functions are detailed. Finally, this article reviews the roles of the ECM and the immune system in islet transplantation as well as ECM-related cytokines and their influence on the ECM and immune cells.

Young porcine endocrine pancreatic islets cultured in fibrin and alginate gels show improved resistance towards human monocytes.

AIM: To investigate the protective function of alginate and fibrin gels used to embed porcine endocrine pancreatic islets towards human monocytes. METHODS: Groups of 200 islet equivalents from young pigs were embedded in either a fibrin or in an alginate gel, and as a control seeded in tissue culture polystyrene (TCPS) well plates. The islet cultures were incubated with 2×10(5) human monocytes for 24h. In addition, both islets and monocytes were separately cultured in TCPS, fibrin and alginate. Islet morphology, viability and function were investigated as well as the secretion of cytokines TNFα, IL-6, and IL-1ß. RESULTS: When freely-floating in TCPS, non-encapsulated islets were surrounded by monocytes and started to disperse after 24h. In fibrin, monocytes could be found in close proximity to embedded islets, indicating monocyte migration through the gel. In contrast, after 24h, few monocytes were found close to islets in alginate. Immunofluorescence staining and manual counting showed that integrin expression was higher in fibrin-embedded islet cultures. A TUNEL assay revealed elevated numbers of apoptotic cells for islets in TCPS wells compared to fibrin and alginate cultures. Insulin secretion was higher with islets embedded in fibrin and alginate when compared to non-encapsulated islets. TNFα, IL-6 and IL-1ß were found in high concentrations in the media of co-cultures and monocyte mono-culture in fibrin. CONCLUSION: Both alginate and fibrin provide key structural support and offer some protection for the islets towards human monocytes. Fibrin itself triggers the cytokine secretion from monocytes.

Composition and organization of the pancreatic extracellular matrix by combined methods of immunohistochemistry, proteomics and scanning electron microscopy.

The epidemic expansion of diabetes is a major concern of public health. A promising treatment is the transplantation of islets of Langerhans isolated from the whole pancreas but the yields of islets isolation and the rates of successful engraftments still have to be improved to make this therapy effective. The extracellular matrix (ECM) of the pancreatic tissue is partially lost during the isolation process and a comprehensive knowledge of the pancreatic ECM composition and organization could identify targets to improve islets isolation and transplantation or highlight new therapeutics for pancreatic diseases. The organization, composition and three-dimensional architecture of the pancreatic ECM were analysed in mouse and pig by three different techniques. Laminin α-4 and ß-2 chains are localized by immunohistochemistry in the exocrine tissue and inside islets of mouse pancreas but not around islets that are surrounded by an ECM made of collagen type IV and type V. Collagen type I, III, and VI were identified by proteomics as specific constituents of the pig pancreatic ECM along with the low-abundance isoforms α3(IV) α4(IV) α5(IV) and α1(V) α2(V) α3(V) of collagen type IV and type V respectively. The three-dimensional ECM architecture is analysed on decellularized mouse pancreas by scanning electron microscopy and is organized in honeycomb structures made of thin ECM fibers assembled in thicker bundles. The combination of immunohistochemistry, proteomics and scanning electron microscopy gives complementary perspective on the pancreatic ECM composition and organization. It represents a valuable toolbox for deeper investigations of ECMs and proposes clues in tissue engineering of the pancreas.

Lipid uptake in synthetic vascular prostheses explanted from humans.

Previous in vivo studies in humans and dogs have revealed an atherosclerosis-like phenomenon in which lipid penetration within arterial prosthesis wall was observed. The primary goal of the present study was therefore to investigate the occurrence of this lipid retention in ePTFE prostheses implanted in humans and therefore identify potential risk factors related to this phenomenon. Lipid uptake in 367 ePTFE microporous vascular prostheses explanted from humans was studied using Fourier transform infrared spectroscopy. The assignment of the infrared absorption features clearly revealed the presence of strongly bonded unsaturated fatty acids to the microporous structure of the prostheses. A one-way ANOVA statistical analysis showed that the lipid uptake in the synthetic vascular prostheses depended on the duration of implantation of the prosthesis and on the sex of the patient. A two-way ANOVA showed that a relationship existed between the estimated lipid uptake and the internal diameter of the prosthesis. These results confirm that the lipid uptake phenomenon depends on some clinical factors related either to the patients or to the prostheses' morphological parameters.

Scaffold vascularization: a challenge for three-dimensional tissue engineering.

The prevalent challenge facing tissue engineering today is the lack of adequate vascularization to support the growth, function, and viability of tissue substitutes that require blood vessel supply. Researchers rely on the increasing knowledge of angiogenic and vasculogenic processes to stimulate vascular network formation within three-dimensional tissue constructs. These processes are mainly endothelial cell-regulated, although in the context of tissue engineering, specific interactions with scaffold materials, growth factors and other cell types may require in vitro vascularization schemes to be altered accordingly. To better mimic the complete in vivo environment, increasing attention is given to the integration of co-cultures and mechanical conditioning in bioreactors. Such approaches show great promise for the enhancement of the functionality and clinical applicability of tissue engineering constructs. This paper reviews some scaffold materials used in tissue engineering and the effect of their properties on the vascularization process. Also, it specifically addresses the pivotal role of biomaterials vascularization in tissue engineering applications, along with the effect of angiogenic factors and adhesive molecules on angiogenesis. Assays and markers of angiogenesis are also outlined. One section highlights the need for bioreactor cultures and mechanical conditioning in controlling endothelial cell responses. Finally, we conclude with a brief section on the effects of oxygen concentration and hypoxia over microvessel formation.

A continuous and pulsatile flow circulation system for evaluation of cardiovascular devices.

The design of a nonpulsatile and pulsatile system using a centrifugal pump is presented. To induce a pulsatile flow with a centrifugal pump, an independent pneumatically driven unit provided flow patterns over a wide range of frequencies and amplitudes. The pulsatile flow was generated by the axial displacement of a cylinder that periodically compressed the flexible conduit that is connected to the pump. The system can accommodate flow rates up to 6,000 ml/min and transmural pressures up to 500 mm Hg and is capable of maintaining the pressure at a constant value. This circuit produced reproducible pressure waves having a frequency up to 4 Hz. The periodicity of the transmural pressure between 80 and 180 mm Hg was similar to the pressure wave propagation observed in peripheral circulation. Capable of adequately reproducing continuous and pulsatile flow, the apparatus is therefore versatile to allow in vitro evaluation of cardiovascular devices.

Lipid uptake across the wall of an expanded polytetrafluoroethylene vascular graft.

Previous studies have shown that vascular grafts were prone to inducing an atherosclerosis-like phenomenon, thus possibly jeopardizing their performance. Furthermore, lipid retention, observed in most synthetic arterial prostheses explanted from humans, appears to have an important role in the progression of this atherosclerotic process, therefore hindering the healing process and neo-intima formation of these synthetic conduits. The current study examined lipid concentration profiles across prosthesis membranes exposed to lipid dispersion under various transmural pressures, flow rates, and durations of exposure. It was demonstrated that the lipids rapidly permeated the prosthesis membrane, as lipid advection increased to a maximum, then steadily decreased until the membrane became completely impermeable to the fluid. The concentration of lipids within the grafts was monitored using FT-IR microspectroscopy, then correlated as a function of time in order to evaluate the mass transfer coefficients and lipid saturation concentration. Lipid sorption, as a function of time, was described by a mechanism taking into account two first-order kinetic models. The lipids were first rapidly adsorbed onto the Teflon(R), potentially influenced by the strong affinity of these lipids for the highly hydrophobic polytetrafluoroethylene polymer. This affinity then enhanced the germination of the lipid deposits that filled in the prosthesis wall. For lipid retention as a function of the transmural pressure and flow rate, no clear tendency was established.

Lipid uptake in expanded polytetrafluoroethylene vascular grafts.

PURPOSE: The mechanisms of vascular prosthesis failure are reported to be associated, in part, with an atherosclerotic degenerative process that is related to an abnormal lipid infiltration. The lipid uptake in expanded polytetrafluoroethylene (ePTFE) vascular grafts was reproduced in vitro, and the effect of time on the permeability of these prostheses was studied. METHODS: Water permeability tests were carried out under dynamic flow conditions at various hydrostatic pressures. Lipid uptake was simulated by circulating a phosphatidylcholine suspension inside an expanded Teflon prosthesis under pulsatile or continuous transmural pressure ranging between 80 mm Hg and 180 mm Hg, at a flow rate of 500 mL/min and 2000 mL/min, for a duration ranging from 2 hours to 1 month. RESULTS: Water permeability tests indicated that under hydrostatic pressures of 180 mm Hg and 300 mm Hg, water percolated through the prosthesis wall after an exposure of 720 minutes and 75 minutes, respectively. After exposing the prostheses to the lipid dispersion under the various flow conditions, the fluid convection through the wall occurred. Preferential convection pathways with a constant periodicity were observed across the length of each prosthesis and were, therefore, associated with regularly spaced perforations depicted in the structure of the devices. Phospholipids gradually agglomerated within the prosthesis wall, allowing a restrictive molecular mobility. Infrared spectroscopy results indicated that the lipid uptake depended on the transmural pressure and time of exposure. CONCLUSION: The occurrence of the membrane permeability may be associated with the dilatation and plastic deformation of the prosthesis. Lipid uptake occurs in ePTFE grafts after an aggressive kinetic process.

Commercial polyurethanes: the potential influence of auxiliary chemicals on the biodegradation process.

This investigation elucidates some aspects of auxiliary chemicals on the biodegradation of two commercial polyurethanes (Pellethane and Corethane). The materials were incubated for 28 days with cholesterol esterase and/or with phosphatidylcholine. Extraction studies were carried out on the two materials, using different solvents, chosen on the basis of solvent polarity. FT-IR spectra for the extracted materials indicated the presence of poly(methylene)n oxide moities, silicone oil, bis-ethylene-stearamide, aromatic moities, and alkyd-urea compounds in Pellethane. Corethane materials were shown to contain some fatty acids, hydrocarbon waxes, ester-based species, and chlorinated compounds. Analysis of incubation solutions by high performance liquid chromatography failed to isolate methylene dianiline (MDA) or any of its derivatives from the various polymer incubation solutions. However, a methanol extract of Corethane samples that were incubated for 28 days in cholesterol esterase did show the presence of MDA. The absence of MDA in the Pellethane methanol extracted samples may reflect the differences in surface additives found for this material versus the Corethane. FT-IR/ATR analysis of polymer surfaces exposed to cholesterol esterase/phospholipids mixture showed that there was an increase in the uptake of phospholipids over samples that were incubated in phospholipid dispersion alone. The results of this study show that some of the auxiliary chemicals found in commercial polyurethanes may hinder the specific release of hydrolytic degradation products and delay polymer degradation. However, it should be recognized that the surface layer containing these compounds is susceptible to change following the interaction between the polyurethane-based devices and elements of the host environment (i.e. lipids, enzymes, etc.). Hence, recognition and identification of these changes will ultimately be important in assessing a commercial polymer's blood compatibility characteristics.

Modeling lipid uptake in expanded polytetrafluoroethylene vascular prostheses and its effects on mechanical properties.

The radial transport across the wall of expanded polytetrafluoroethylene (ePTFE) arterial prostheses has a significant effect on lipid uptake observed in prostheses implanted in humans, which has been postulated to be one of the causes associated with implant failure. The goal of this study was to stimulate radial transport on a lipidic dispersion across the wall of an ePTFE prosthesis and investigate its effects on the circumferential mechanical properties of the prosthesis. An in vitro model was developed to simulate the lipidic radial transport across the wall. Lipids contained in a phosphatidylcholine dispersion were used as the transported molecules. Lipid concentration profiles were obtained after exposing commercial ePTFE prostheses to various transmural pressure and/or lipidic concentration gradients. Phospholipids gradually accumulated up to the external reinforcing wrap of the prosthesis, which clearly acted as a rigid barrier against lipid infiltration. Tensile tests performed on the virgin samples showed that the wrap was much more rigid than the microporous part of the prosthesis. After the lipid simulation, the rigidity of the wrap decreased with respect to what was observed for the virgin prosthesis. Finally, some clinical implications of this phenomena are discussed.

Lipid concentration profile across the wall of pseudoatherosclerotic synthetic arterial prostheses using FTIR microspectroscopy.

FTIR microscopy is a versatile technique successfully used to probe the subcellular chemical composition of atherosclerostic arterial walls. To design new vascular substitutes that resist lipid uptake (the major cause of the phenomenon referred to as atherosclerosis-like), identifying and understanding lipid distribution within the pseudoatherosclerosed arterial prostheses is of prime importance. Until now, the amount of lipids present within arterial prostheses that had been explanted from either animals (during in vivo trials) or humans (after the failure of vascular grafts) or had been submitted to in vitro investigations could only be measured through the use of histological techniques or radioactive labeling methods. We present here a novel method to quantitatively measure the lipid concentration profile within the wall of arterial prostheses by means of Fourier transform infrared microspectroscopy. Essentially, prostheses are fixed in a 1% osmium tetraoxide aqueous solution under vacuum and radially cut with a 5-micron thickness with a microtome. The sections are then placed onto BaF2 windows and observed with a microscope attached to a FTIR spectrometer with a 30 microns x 50 microns sampling area. The lipid concentration profile is obtained by scanning the prosthesis wall from the inner to the outer surface and reporting the corresponding integrated absorbance between 2700 and 3100 cm(-1) against a calibration curve. The application of this technique constitutes the first quantitative measurement of the concentration of biological molecules within the wall of artificial arterial substitute.