Chitosan-based hydrogels for developing a small-diameter vascular graft: in vitro and in vivo evaluation.

AIMS: Vascular grafts made of synthetic polymers perform poorly in small-diameter applications (cardiac and peripheral bypass). Chitosan is a biocompatible natural polymer that can provide a novel biological scaffold for tissue engineering development. The goal of this study was to demonstrate the biocompatibility of a novel chitosan preparation in vitro and in vivo, and to assess its potential as a scaffold for vascular applications. METHODS AND RESULTS: A series of experiments of increasing complexity, ranging from in vitro biocompatibility and hemocompatibility tests to in vivo studies in small and large animals (rats and sheep), was performed to provide a comprehensive analysis of chitosan hydrogels' biological properties. In vitro studies established that: (i) chitosan supported human endothelial progenitor cells adhesion, proliferation and resistance to physiological shear stress; (ii) chitosan did not activate platelets, the complement system, or the intrinsic coagulation pathway. In vivo results showed: (iii) no resorption of chitosan and no chronic inflammation at 60 days in a rat heterotopic implantation model (magnetic resonance imaging and histology); (iv) no flow obstruction (Doppler ultrasound) and no thrombus formation (histology and scanning electron microscopy) at 2 h after a carotid arteriotomy repair with chitosan patches in sheep. Finally, two chitosan tubes were implanted as carotid interposition grafts for 3 days in sheep showing that chitosan was strong enough to be sutured, to withstand arterial pressure, and no flow obstruction was observed through this short period. CONCLUSION: Chitosan-based hydrogels displayed promising in vitro biocompatibility and hemocompatibility properties as well as in vivo short-term performance.

Whatever their differentiation status, human progenitor derived - or mature - endothelial cells induce osteoblastic differentiation of bone marrow stromal cells.

Association of the bone-forming osteoblasts (OBs) and vascular endothelial cells (ECs) into a biomaterial composite provides a live bone graft substitute that can repair the bone defect when implanted. An intimate functional relationship exists between these cell types. This communication is crucial to the coordinated cell behaviour necessary for bone development and remodelling. Previous studies have shown that direct co-culture of primary human osteoprogenitors (HOPs) with primary human umbilical vein endothelial cells (HUVECs) stimulates HOPs differentiation and induces tubular-like networks. The present work aims to test the use of human bone marrow stromal cells (HBMSCs) co-cultured with human endothelial progenitor cells in order to assess whether progenitor-derived ECs (PDECs) could support osteoblastic differentiation as mature ECs do. Indeed, data generated from the literature by different laboratories considering these co-culture systems appear difficult to compare. Monocultures of HUVECs, HOPs, HBMSCs (in a non-orientated lineage), PDECs (from cord blood) were used as controls and four combinations of co-cultures were undertaken: HBMSCs-PDECs, HBMSCs-HUVECs, HOPs-PDECs, HOPs-HUVECs with ECs (mature or progenitor) for 6 h to 7 days. At the end of the chosen co-culture time, intracellular alkaline phosphatase (ALP) activity was detected in HOPs and HBMSCs and quantified in cell extracts. Quantitative real-time polymerase chain reaction (qPCR) of ALP was performed over time and vascular endothelial growth factor (VEGF) was measured. After 21 days, calcium deposition was observed, comparing mono- and co-cultures. We confirm that ECs induce osteoblastic differentiation of mesenchymal stem cells in vitro. Moreover, HUVECs can be replaced by PDECs, the latter being of great interest in tissue engineering.

Polyelectrolyte multilayer films allow seeded human progenitor-derived endothelial cells to remain functional under shear stress in vitro.

There is considerable interest in making multilayer films for various applications, among which are cell contacting biomaterials, allowing new opportunities to prepare functionalized biomaterials. In this study we have explored the capability of poly(sodium-4-styrene sulfonate)/poly(allylamine hydrochloride) polyelectrolyte multilayer films (PMFs) as functional coatings for human progenitor-derived endothelial cells (PDECs), since the latter are a potential source of endothelial-type cells to be used in bioartificial vascular substitutes. We performed investigations with PDECs derived from peripheral blood and characterized as endothelial cells. After forming a confluent monolayer on PMFs they were exposed to laminar pulsatile physiological shear stress. We investigated whether PDECs were able to withstand shear stress and to respond at the mRNA (microarray analysis) and protein levels (thrombomodulin and tissue factor functional activity), in comparison with collagen I and fibrin glue used as controls. After shear stress the PDECs remained spread on the substrates, with a resulting increase in the number of expressed genes. Considering the functional significance of our findings for the regulation of coagulation and fibrinolytic factors, mRNA tissue plasminogen activator and thrombomodulin, profibrinolytic and thrombin inhibiting respectively, were overexpressed in PDECs after 6h shear stress. von Willebrand factor showed down-regulation, while tissue factor was up-regulated. We can speculate that PMFs could favour anti-thrombogenic activity by PDECs because activated protein C generation, measuring thrombomodulin activity, was particularly high on PMFs, but unchanged after 6h shear stress. Thus, PMFs could represent suitable coatings able to provide functional surfaces for endothelialization with PDECs.

Ecology of lactobacilli in the oral cavity: a review of literature.

Lactobacilli appear in the oral cavity during the first years of a child's life. Their presence depends on numerous factors such as the presence of ecological niches e.g. natural anfractuosities of the teeth.A strong correlation has been established between the saliva Lactobacillus count and dental caries, the higher the DMF index, the higher the number of children harbouring a high Lactobacillus count.Among children, the presence of lactobacilli in coronal caries is incontestable. Among adults, lactobacilli are found in root caries. Since 1999, taxonomical revisions make it difficult to interpret the results obtained in the numerous previous studies carried out on the identification of oral lactobacilli, but whatever the sampling method or the identification technique, the carious site or the age of sampled subjects, most species belong to the Lactobacillus casei group.This is important because if a specific correlation can be found between few species of lactobacilli and caries a better understanding of their properties could allow the development of new tools for prevention.