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Digital archive of theses discussed at the University of Pisa


Thesis etd-10042010-111831

Thesis type
Tesi di dottorato di ricerca
Thesis title
Development of bioengineered matrices for the release of angiogenesis-promoting cells and ischemic tissue vascularization
Academic discipline
Course of study
tutor Prof. Balbarini, Alberto
  • biomaterials
  • endothelial progenitor cells
  • fibrin
  • peptide amphiphile
  • scaffolds
  • tissue engineering
Graduation session start date
Release date
Background: Endothelial progenitor cells (EPCs) contribute to endothelial cell regeneration of injured vessels as well as neovascularization of ischemic lesions. EPC in situ release and retention offers new possibilities for cardiovascular tissue engineering. For this reason it is important to use biocompatible and biodegradable scaffolds mimicking the structure and biological function of native extracellular matrix (ECM). Fibrin and peptide amphiphile are examples of such biomaterials. Fibrin is a natural biocompatible biopolymer, obtained by enzymatic polymerization of fibrinogen catalyzed by thrombin. Fibrin is suitable for healing and angiogenesis, but has also properties for tissue engineering application. Peptide amphiphiles (PA) are self-assembling biocompatible molecules with the potential of forming bioactive hydrogels composed of nanofibers, when mixed with opposite charged solution. They consist of a short hydrophilic peptide segment covalently bonded to an hydrophobic fatty acid chain. PA can include sequences present in ECM, such as arginine-glycine-aspartic acid (RGD).
Aim: Aim of this project to study the interaction between EPCs and natural (fibrin) or synthetic (PA) biomaterials, for potential therapeutic application. Biomaterials were used to induce EPC growth, differentiation and functional activity. The results were compared with those obtained with the traditional substrate, fibronectin.
Materials and Methods: 1×106 cell/cm2 peripheral blood mononuclear cells of healthy donors were cultured for 1 week on fibronectin in an endothelial medium with 5% FBS and growth factors to obtain EPCs.
Fibrin was characterized for stiffness, capability to sustain cell growth and fiber diameter and density at different fibrinogen-thrombin ratios. PA structure contained RGD, 8 aminoacids and an alkyl tail of 16 carbons. PA gel formation was evaluated using pH change or solutions containing opposite charged ions. Gels were characterized for their stiffness. Fibrin and PA gel ultrastructure was investigated by scanning electron microscopy (SEM), atomic force microscopy (AFM) and cryogenic SEM (CRYO-SEM).
Mononuclear cells (1×106 cell/cm2) were seeded on the fibrin composition that gave the best results with the preliminary experiments. PA gels were formed by mixing the aqueous PA solution at different concentrations (1%-2%) with the growth medium supplemented of CaCl2. (10-20 mM). Mononuclear cells (1×106 cell/cm2) were seeded either on the surface (2-D) or inside PA gel (3-D) and cultured for 1 week to obtain EPC. Cell viability and growth were evaluated by confocal microscopy (Calcein AM staining) and conversion of a tetrazolium salt (WST-1 assay). The expression of endothelial markers (CD31, KDR, vWF, Ve-Cadherin) was assessed by flow cytometry and confocal microscopy. Cell morphology was assessed also by confocal microscopy (vimentin staining). Stem cell markers (OCT 3/4 and NANOG) expression was evaluated by quantitative Real Time RT-PCR and by confocal microscopy. EPC angiogenic function was evaluated by their participation to tubule networks formed by human umbilical vein endothelial cells (HUVECs) on Matrigel. EPC release of 50 cytokines involved in inflammatory process and chemotaxis was evaluated by a multiplexable bead assay (Bio-Plex). EPC obtained on fibronectin were used as a control.
Results: Peripheral blood mononuclear cells culture for 1 week in endothelial growth medium on fibronectin resulted in a cell population with the phenotype of “early” EPCs, as shown by markers expression, proliferation data and cell participation to tubule networks.
Increased fibrinogen concentration significantly enhanced fibrin stiffness, decreased fiber diameter and reduced cell growth. 9 mg/ml fibrinogen and 25 U/ml thrombin resulted the best ratio. Fibrin showed a nanometric fibrous structure and a porous network, with micropores of different size. Cell viability was significantly higher on fibrin as compared to fibronectin. Even though no significant difference was observed in the expression of endothelial markers, culture on fibrin elicited a marked induction of stem cells markers OCT 3/4 and NANOG. The in vitro angiogenesis assay on Matrigel showed that EPCs grown on fibrin retain angiogenesis capability as EPCs grown on fibronectin, but HUVEC ability to form tubules was significantly increased when co-plated with EPCs detached from fibrin. A significant higher release of cytokines involved in cell recruitment (IL-16, PDGF-BB, MIF, SDF-1, HGF, IP-10, MIG) was produced by EPC grown on fibrin.
PA had a native pH of 4.0 but gained solubility when pH=7.4 was reached. PA spontaneously forms nanofibers that do not assemble into gels. With PA concentration ≥ 1%, a gel was obtained by pH change or by adding solutions containing CaCl2. AFM analysis showed the presence of networks of nanofibers. Morphological analysis of PA revealed the presence of 3-D network of nanofibers, assembled in a reticulate structure. AFM showed that gel stiffness is not affected by the initial PA concentration, but is proportional to PA/CaCl2 ratio. A significantly higher viability as compared to fibronectin was observed at PA concentration of 1% in the 3-D seeding model. Confocal microscopy showed that cells expressed endothelial markers when seeded on PA. A significant higher release of chemokines involved in cell homing and recruitment (IL-8, MCP-1, VEGF, SCGF-) was observed as compared to cells grown on fibronectin.
Conclusion: Both fibrin and self-assembling PA gels can support peripheral blood early EPC viability, differentiation and angiogenic function. The two biomaterials enhance cell retention and paracrine cytokine release, suggesting their potential application for EPC transplantation.