• scaffold
• renewable resources
• Alginate

The main aim of the modern scientific interdisciplinary field known as Tissue Engineering is to design artificial biocompatible materials to substitute irreversibly damaged tissue of an organs. Its strategy focuses on the creation of hybrid artificial organs to improve the quality of life for many patients. Tissue engineering is finding wide application in liver regeneration. The need to develop new artificial substitutes to use in liver tissue engineering is due to its crucial role in body’s metabolism. The number of functions in the body, including glycogen storage, decomposition of red blood cells, plasma protein synthesis, and detoxification highlight the importance to develop new methodologies that will be able to restore the complete functionality of theorgan damaged by traumatic and pathologic events. Actually, the common procedures for liver failures still remain organ transplantation. However, as demand for donor organs continues to increase beyond their availability, the need for alternative therapies is clear. Several approaches have been proposed including extracorporeal devices. The artificial liver systems represent a temporary bridge to support a patient awaiting for a new organ but the number of hepatocytes required is higher than the real availability of cells. In this respect the need for more effective solutions appear extremely important. The development of biological substitutes based on living cells grown and supported onto polymer matrices seems to be a valid solution.
In the framework of a long-standing activity ongoing at the Bioactive Polymeric Material group at the Department of Chemistry and Industrial Chemistry of the University of Pisa, the present work was aimed both at the development of polymeric scaffolds based on naturally derived materials for cell culturing and particles containing cells and/or tissue specific growth factors to be used in regenerative medicine of liver. In the present PhD thesis the potential of a new type of alginate, namely Ulvan derived from marine renewable resource, to be used in biomedical applications as alternative material to commercial alginate was submitted to investigation. Four different batches of Ulvan with different physical aspect (flakes, powder and laminae), were investigated. The use of natural polymers, or biopolymers, as structural material is known since a while. Polysaccharides are known to show a variability and versatility, associated with their complex structures, not found in other classes of polymers and their gelling ability are increasingly being used in biotechnological applications. The improvement of the stability of soft gels produced by these polysaccharides represented the first research goal. By introduction of physical and chemical crosslinking agents in scaffolds formulation the mechanical stability of these polysaccharides were successfully improved and scaffolds resistant to dissolution in physiological conditions were obtained. The morphological characterization of the prepared constructs, as performed by Scanning Electron Microscopy (SEM), revealed the interconnected porous structure with pores size (100 µm) favourable to cell attachment and proliferation. A careful in vitro investigation of Ulvan cytotoxicity was carried out by using neutral red uptake assay aimed to assess the viability of cells exposed to different polymer concentrations. Quantitative results clearly showed a good cytocompatibility of the investigated Ulvan samples also at high concentration such as 10 mg/ml. These results were further confirmed by morphological investigation performed by using optical microscopy.
The prepared systems were finally submitted to biological evaluation by seeding a hepatoblastoma cell line (HepG2). The gained results revealed not only the mechanical stability of the prepared constructs under physiological conditions but also their capability to sustain cell adhesion and proliferation. The seeded cells indeed formed large aggregates, namely spheroids, maintaining their morphology and viability in the three dimensional structure for all the investigation period.
Tissue engineering of cell loaded beads represent another emerging cellular therapy for liver diseases. The encapsulation of cells into microcapsules has been widely explored by several reasearchers to overcome the problem of immune rejection or allogenic tissue. The ionotropic gelation technique based on the gelation using divalent cation was exploited for the preparation of HepG2 loaded beads. Cells were successfully encapsulated in spherical beads with smooth surface and 700 ±328 μm in size. Quantitative evaluation of cell proliferation performed by mean of WST-1 tetrazolium salt assay revealed that encapsulated cells in alginate beads display a proliferation comparable to the monolayer culture. Cell morphology was assessed by microscopy after treatment with Neutral Red.
With today’s interest in novel renewable chemicals and polymers, the underexploited marine green algae belonging to Ulva species stimulated our interest as source of polysaccharides with innovative structure and functional properties. Their relatively low cost could also promote their use in environmental and pharmaceutical applications. The chemical structure and type of gelling mechanism of these natural polymers may strongly affect the final properties of the polymeric network and therefore their final application. For all these reasons the structure of Ulvan was deeply investigated.
Chemical-physical characterization of Ulvan was carried out in order to determine the molecular weight and the content of neutral sugars, uronic acids and proteins. Molecular weight evaluated by means of Size Exclusion Chromatography (SEC) indicated an Mw value in the range of 50-60 kDa. The content of neutral sugars determined by gas liquid chromatography (GLC) as referred to 2-deoxy-D-galactose commercial standard, displayed clear abundance of rhamnose with respect to the other constituent sugars. Uronic acid content in Ulvan was assessed using a colorimetric assay based on 3-hydroxybipheny and glucuronic acid as reference standard Contents of Uronic acid in the range of 10% were detected and the performed analyses confirmed the heterogeneous structure of the Ulvan under investigation. All obtained results on Ulvan composition combined with the results obtained from Ulvan based scaffolds preparation strongly suggest the suitability of this new type of alginic materials for biomedical applications. Further investigation will be aimed at setting up an appropriate technique for Ulvan based beads preparation and the viability evaluation of encapsulated cells.