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Introduction
Tissue Engineering may one day generate organs for transplantation that may be able to treat a variety of debilitating diseases, such as Osteoporosis, Diabetes and Parkinson’s disease. One of the major barriers to realization of this enormous potential is the lack of a renewable source of cells for transplantation. Embryonic stem cells (ESCs) have the potential to provide a source of cells because of their ability to differentiate to all somatic cells and their unlimited proliferative capability. In this work the potential and the challenges associated with the use of ESCs in bone tissue engineering will be discussed. In particular, the use of three-dimensional matrices “scaffolds” seems to be a winning and innovative way to replace extracellular matrix in vitro and to guide tissue regeneration in vivo. This work describes the techniques and design criteria to realize innovative scaffolds for bone tissue engineering (in particular through use of human fetal stem cells) able to induce stem-cells proliferation and differentiation into mature osteoblasts. In recent years several methods of scaffold fabrication have been developed to different aspects of tissue-formation processes. Spatiotemporal control of scaffold biochemical and physical properties has been investigated controlling fundamental molecules and cell biomechanics into scaffolds. Because living cells and tissues are highly complex systems, a central theme in designing tissue engineering scaffolds is to understand how scaffold structures and its properties are correlated to cell and tissue functions.
A key concept in tissue engineering (TE) is use porous 3D scaffolds to provide physical support and a local environment for cells to enable and facilitate tissue development. Since the mid-1980s, 3D scaffolds have evolved to serve complex functions of guiding cellular behavior in different TE applications. Scaffolds can be seeded with embryonic or adult stem cells, progenitor cells, mature differentiated cells, or co-cultures of cells to induce tissue formation in vitro and in vivo. Scaffolds can also be directly implanted in vivo with capabilities to deliver soluble/insoluble biomolecules and temporal/spatial cues to guide the function regeneration in defected tissues and organs. While the specific functions vary with tissue type and clinical need, scaffolds may potentially coordinate biological events at the molecular, cellular, and tissue levels on time and length scales ranging from seconds to weeks and nanometers to centimeters, respectively.
2 A central theme in designing tissue-engineering scaffolds is to understand the correlations between scaffold properties and biological functions. During tissue development, cells constantly decode and release different morphogenetic factors in their surroundings. In response, cells make decisions on how to divide, differentiate, migrate, degrade/produce extracellular matrix (ECM), and orient themselves. Based on our understanding of cell-ECM interactions in native tissues, it should theoretically be possible to design scaffolds able to promote tissue formation through systematic variations in one or more chemical and/or physical properties.
Living cells and tissues, however, are not simple, linear systems. The complex role that ECM structures play in generating cellular and tissue processes can be thought of as a multiband signal to which different cell types may exhibit radically different sensitivities. Moreover, the performance of any scaffold must be evaluated not only in the relatively controlled in vitro environment, but also in the context of the tissue’s physiological functions (under mechanical or electrical stimulation) and ultimately in vivo.
The dynamic superposition of multiple scaffold properties with multiple environmental factors can profoundly impact the outcome of the tissue formation process. Correlations between individual scaffold properties and some aspects of tissue formation (cellular phenotype and concomitant proliferation or ECM production) can be derived, and they have been applied to the optimization of scaffolds designs. From an engineering standpoint, it is desirable that scaffold properties and their regulatory functions be reproducibly integrated within a scaffold design such that tissue formation can be induced and controlled in a stepwise manner in vitro and/or at the site of implantation in vivo.
A recent preferred trend is to combine different materials to produce a composite structure. Composite scaffolds are expected to be physically and biologically superior to single-material based scaffolds, for the properties of a composite may be programmatically varied by mixing different materials in various ratios. Both the composition and the relative ratio of constituent materials can affect bone formation. Hydroxyapatite (HA) has been used as a primary material combined with another material, such as tricalcium phosphate (TCP), PLGA, and chitin, to produce various composite scaffolds. It was reported that scaffolds with different ratios of HA/TCP loaded with MSCs showed different extents of bone formation in vivo. Composites in which the HA/TCP ratio was designed to coordinate scaffold degradation with tissue deposition seemed optimal in promoting the greatest ectopic bone formation.
3 Another recent discovery in scaffolding for bone tissue engineering is the creation of a composite scaffold based on the properties of CNT (Carbon Nanotubes). CNTs offer a natural platform to obtaine composite microfabricated scaffolds thanks to their excellent mechanical and electronic properties, coupled with a good biocompatibility. Three-dimensional PLLA/CNTs composite scaffolds showed higher stiffness that pure three-dimensional PLLA scaffolds, and so they seems to be eligible for bone tissue engineering applications. In this way, the objective of this work is to designe and realize 3-D PLLA/MWCNT composite scaffolds through the use of PAM microfabrication technique. These scaffolds will be tested in vitro with hFOB 1.19 (human fetal osteoblast) cells to induce cell differentiation into mature osteoblast phenotype. In particular, a preliminar mechanical characterization and then a cellular analysis will be performed to show what kind of scaffold (composite or not) could be more suitable for bone tissue engineering applications.
This thesis-work has been developed at California Institute of Technology (Caltech) thanks to a SURF (Summer Undergraduate Research Felloship) program I applied in summer 2009 from June To September. In particular, scaffold mechanical characterization has been performed in department of Aeronautics and Applied Physics with mentor Prof. Chiara Daraio, while cellular analysis has been performed in Broad Centre for Biological Science (Caltech).
