Horia Maniu

Anionic polymers and 10nm Fe3O4@UA wound dressings support human foetal stem cells normal development and exhibit great antimicrobial properties

The aims of this study were the development, characterization and bioevaluation of a novel biocompatible, resorbable and bio-active wound dressing prototype, based on anionic polymers (sodium alginate--AlgNa, carboximethylcellulose--CMC) and magnetic nanoparticles loaded with usnic acid (Fe₃O₄@UA). The antimicrobial activity was tested against Staphylococcus aureus grown in biofilms. The biocompatibility testing model included an endothelial cell line from human umbilical vein and human foetal progenitor cells derived from the amniotic fluid, that express a wide spectrum of surface molecules involved in different vascular functions and inflammatory response, and may be used as skin regenerative support. The obtained results demonstrated that CMC/Fe₃O₄@UA and AlgNa/Fe₃O₄@UA are exhibiting structural and functional properties that recommend them for further applications in the biomedical field. They could be used alone or coated with different bio-active compounds, such as Fe₃O₄@UA, for the development of novel, multifunctional porous materials used in tissues regeneration, as antimicrobial substances releasing devices, providing also a mechanical support for the eukaryotic cells adhesion, and exhibiting the advantage of low cytotoxicity on human progenitor cells. The great antimicrobial properties exhibited by the newly synthesized nano-bioactive coatings are recommending them as successful candidates for improving the implanted devices surfaces used in regenerative medicine.

Histone deacetylase (HDAC) inhibitors down-regulate endothelial lineage commitment of umbilical cord blood derived endothelial progenitor cells.

To test the involvement of histone deacetylases (HDACs) activity in endothelial lineage progression, we investigated the effects of HDAC inhibitors on endothelial progenitors cells (EPCs) derived from umbilical cord blood (UCB). Adherent EPCs, that expressed the endothelial marker proteins (PCAM-1, CD105, CD133, and VEGFR2) revealed by flow cytometry were treated with three HDAC inhibitors: Butyrate (BuA), Trichostatin A (TSA), and Valproic acid (VPA). RT-PCR assay showed that HDAC inhibitors down-regulated the expression of endothelial genes such as VE-cadherin, CD133, CXCR4 and Tie-2. Furthermore, flow cytometry analysis illustrated that HDAC inhibitors selectively reduce the expression of VEGFR2, CD117, VE-cadherin, and ICAM-1, whereas the expression of CD34 and CD45 remained unchanged, demonstrating that HDAC is involved in endothelial differentiation of progenitor cells. Real-Time PCR demonstrated that TSA down-regulated telomerase activity probably via suppression of hTERT expression, suggesting that HDAC inhibitor decreased cell proliferation. Cell motility was also decreased after treatment with HDAC inhibitors as shown by wound-healing assay. The balance of acethylation/deacethylation kept in control by the activity of HAT (histone acetyltransferases)/HDAC enzymes play an important role in differentiation of stem cells by regulating proliferation and endothelial lineage commitment.

Direct contact of umbilical cord blood endothelial progenitors with living cardiac tissue is a requirement for vascular tube-like structures formation.

The umbilical cord blood derived endothelial progenitor cells (EPCs) contribute to vascular regeneration in experimental models of ischaemia. However, their ability to participate in cardiovascular tissue restoration has not been elucidated yet. We employed a novel coculture system to investigate whether human EPCs have the capacity to integrate into living and ischaemic cardiac tissue, and participate to neovascularization. EPCs were cocultured with either living or ischaemic murine embryonic ventricular slices, in the presence or absence of a pro‐angiogenic growth factor cocktail consisting of VEGF, IGF‐1, EGF and bFGF. Tracking of EPCs within the cocultures was performed by cell transfection with green fluorescent protein or by immunostaining performed with anti‐human vWF, CD31, nuclei and mitochondria antibodies. EPCs generated vascular tube‐like structures in direct contact with the living ventricular slices. Furthermore, the pro‐angiogenic growth factor cocktail reduced significantly tubes formation. Coculture of EPCs with the living ventricular slices in a transwell system did not lead to vascular tube‐like structures formation, demonstrating that the direct contact is necessary and that the soluble factors secreted by the living slices were not sufficient for their induction. No vascular tubes were formed when EPCs were cocultured with ischaemic ventricular slices, even in the presence of the pro‐angiogenic cocktail. In conclusion, EPCs form vascular tube‐like structures in contact with living cardiac tissue and the direct cell‐to‐cell interaction is a prerequisite for their induction. Understanding the cardiac niche and micro‐environmental interactions that regulate EPCs integration and neovascularization are essential for applying these cells to cardiovascular regeneration.

Integration properties of Wharton's jelly-derived novel mesenchymal stem cells into ventricular slices of murine hearts.

Wharton’s jelly (WJ) is a rich source of multiple-lineage differentiating cells, recently proposed for cell replacement therapy. However, their ability to integrate into the cardiac tissue has not been elucidated, yet. We employed in vitro cardiac transplantation models to investigate the capacity of a novel population of human WJ-derived mesenchymal stem cells (nMSCs) to integrate into both living and ischemic cardiac tissue. NMSCs were characterized for the expression of stem/progenitor cell genes and proteins, as well as for multi-lineage differentiation potential. To assess their integration properties, nMSCs were cocultured with either living or ischemic embryonic murine ventricular slices. Immunohistochemical analyses were performed on cryosections of cocultured preparations to allow human cells tracking within the cocultures. Results showed that nMSCs shared MSC and endothelial colony-forming cell characteristics at gene, protein, and functional levels. NMSCs were markedly chemoattracted towards the ventricular slices, integrating robustly into the depth of both living and ischemic cardiac tissue. In conclusion, the functional ability of WJ-derived cells to populate the cardiac tissue could be validated in vitro. The transplantation models described could be further used to depict the mechanisms of WJ-derived cells integration into the cardiac tissue, contributing to optimization of reliable cell therapies for cardiac repair.

Bioactive ZnO Coatings Deposited by MAPLE—An Appropriate Strategy to Produce Efficient Anti-Biofilm Surfaces

Deposition of bioactive coatings composed of zinc oxide, cyclodextrin and cefepime (ZnO/CD/Cfp) was performed by the Matrix Assisted Pulsed Laser Evaporation (MAPLE) technique. The obtained nanostructures were characterized by X-ray diffraction, IR microscopy and scanning electron microscopy. The efficient release of cefepime was correlated with an increased anti-biofilm activity of ZnO/CD/Cfp composites. In vitro and in vivo tests have revealed a good biocompatibility of ZnO/CD/Cfp coatings, which recommend them as competitive candidates for the development of antimicrobial surfaces with biomedical applications. The release of the fourth generation cephalosporin Cfp in a biologically active form from the ZnO matrix could help preventing the bacterial adhesion and the subsequent colonization and biofilm development on various surfaces, and thus decreasing the risk of biofilm-related infections.

Recellularization potential assessment of Wharton's Jelly-derived endothelial progenitor cells using a human fetal vascular tissue model

Mesenchymal stem cells isolated from Wharton’s Jelly have demonstrated an excellent differentiation potential into the endothelial lineage. We hypothesize that endothelial progenitor cells differentiated from Wharton’s Jelly-derived mesenchymal stem cells have the potential to repopulate a decellularized vascular bed employed as a biological scaffold. For this purpose, we aimed at investigating the behavior of the endothelial progenitor cells in the decellularized matrix and their potential to repopulate decellularized human vascular tissue. Our main objectives were to differentiate Wharton’s Jelly-derived mesenchymal stem cells into endothelial progenitor cells and to obtain a human vascular tissue slice experimental model using the umbilical cord arteries. We employed a decellularization method using enzymatic treatment of the umbilical cord arteries and a recellularization method with the endothelial progenitor cells differentiated from Wharton’s Jelly mesenchymal cells in a co-culture system, in order to investigate our hypothesis. The cellular integration within the biological scaffold was determined by using flow cytometry analysis and confirmed by visualization of histological staining as well as fluorescence microscopy. The morphological observations of the recellularized scaffolds revealed the presence of endothelial progenitor cells within the decellularized tissue slices, displaying no degradation of the scaffold’s extracellular matrix. The flow cytometry analysis revealed the presence of Wharton’s Jelly-derived endothelial progenitor cells population in the decellularized fetal blood vessel scaffold after recellularization. In conclusion, our results have shown that an in vitro human vascular tissue slice experimental model using decellularized human fetal arteries is able to sustain an adequate scaffold for cellular implants.

Poly(lactic-co-glycolic) acid/chitosan microsphere thin films functionalized with Cinnamomi aetheroleum and magnetite nanoparticles for preventing the microbial colonization of medical surfaces

Poly(lactic-co-glycolic) acid/chitosan microsphere coatings containing Cinnamomi aetheroleum-functionalized magnetite nanoparticles (Fe3O4@CA) were deposited by matrix-assisted pulsed laser evaporation in order to improve the surface resistance to microbial colonization. The obtained surface proved a very good biocompatibility and demonstrates that bacterial colonization is impaired on the nanomodified bioactive surfaces and also Staphylococcus aureus biofilm formation is significantly altered.Graphical Abstract

Effects of plant lectin and extracts on adhesion molecules of endothelial progenitors

Promise of cell therapy has advanced the use of adult stem cells towards the development of novel approaches to promote regeneration of injured endothelium. The aim of this study was to stimulate endothelial progenitor cells (EPCs) with lectin isolated from Solanum tuberosum (potato) shoot and Calendula officinalis (marigold) extracts, in order to increase EPCs proliferation and gene expression of molecules with roles in chemotaxis and adhesion for a better attachment to injured vascular tissue. EPCs were differentiated from umbilical cord blood-derived mononuclear cells and characterized by light microscopy, flow cytometry, and vascular tube-like structures formation on Matrigel. Cell proliferation was determined by MTS assay, and gene expression of molecules involved in EPCs adhesion (VCAM-1, VE-cadherin, ICAM-1, PECAM-1, P-selectin) and chemotaxis was determined (CXCR4, Tie-2) by RT-PCR. For the assessment of cell motility, wound-healing assay was employed. Both potato shoot lectin and marigold extracts stimulated EPCs proliferation in a concentration dependent manner and were able to increase expression of adhesion and chemotactic molecules. Marigold flower extract proved to be more efficient. This study demonstrates the usefulness of potato lectin and marigold extracts to increase EPCs proliferation and modulate gene expression of chemotactic and adhesion molecules, which may facilitate EPCs attachment to injured endothelium.

Development of Scaffolds for Vascular Tissue Engineering: Biomaterial Mediated Neovascularization.

Cardiovascular diseases remain the leading cause of mortality or disabled quality of life for people over the world. The necessity of neovascularization is essential for re-establishing the tissue functions after a major lesion that occurs in patients with cardiovascular disorders, such as ischemia, atherosclerosis, diabetes, peripheral vascular disease and burn wounds. This review focuses on the recent data regarding the polymers and scaffolds that are used for improving neovascularization with emphasis on the biocompatibility and mechanisms involved in stem cells proliferation, migration, adherence, differentiation and organization in vascular networks. The newly emerging techniques used in conjugation of synthetic polymers with polysaccharides or proteins attempt to improve the biocompatibility of scaffolds, but the complex structures of blood vessels make their construction to remain a major challenge for the vascular tissue engineering.

Molecular and Cellular Biology of the Right Heart

The present chapter attempts a detailed yet comprehensive account of the exceedingly complex molecular events involved in heart development, including description of the main differentiation signaling pathways, transcription factors, enhancers and gene regulatory networks, with a special emphasis on applying this continuously enlarging body of modern scientific knowledge to the field of stem cell differentiation into cardiomyocytes for diagnostic and therapeutic applications. MicroRNAs are also emerging as an important area of discovery aiming to revolutionize current therapies in cardiology, therefore we describe their involvement in cardiogenesis, proliferation/apoptosis, angiogenesis, fibrosis and hypertrophy. After reviewing the architectural organization, ultrastructure details and functional compartments of cardiomyocytes derived from in vitro and in vivo studies, we focus on molecular mechanisms involved in cardiac hypertrophy and fibrosis, particularly on calcium signaling events triggering these excessive adaptive responses. Further, we stress the embryogenetic and molecular differences between the right and left ventricle, as well as their pathophysiology specificities and clinical consequences.