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LABORATORY OF: Stem Cell Bioengineering
CONTACT PERSON: Dr Silvia Scaglione (University of Genova)
Phone +39 010 5737505 E-mail: Silvia77@dist.unige.it

Description of Laboratory and Expertise:

The main research activities of the laboratory are focused on tissue engineering field and in particular on generation of 3D tissues to be used as grafts for replacement of damaged organsas in vitro model systems of tissue development and function. In particular, the laboratory aims to plan, develop and test 3D systems (bioreactors and biomaterials) where adult progenitors/stem cells may grow under suitable physical chemical stimuli and induce an in vivo neo-bone formation. The laboratory is involved in several projects funded by MIUR related to the area of tissue engineering.

Abstract of Activities:

The research activity focuses on the generation of bone grafts/substitutes, based on autologous cells and 3D scaffolds. In particular, we aim at (i) identifying the best internal structures and chemical compositions of the 3D scaffolds to be used as skeletal (bone, cartilage, ligaments) grafts/substitutes, and (ii) planning and developing specific bioreactors systems (2D-3D) where culture progenitor cells are cultured into biomaterials under monitored physical stimuli (perfusion, shear stress, mechanical stimuli).
Different bioreactor systems for an efficient seeding and culture of cells into 3D scaffolds under automated and controlled conditions have been developed, used and continuously updated to engineer skeletal tissues.
Moreover, we propose to identify genes involved in the mechanism of proliferation and differentiation of human MSC under different experimental conditions by studying their gene expression profile through the micro-array technique. Advanced technologies and knowledge related to production and treatment of gene expression micro-array distributed data have been implemented, in collaboration with the Bio-Lab laboratory of University of Genova and the genomic unit of ABC (Pfeffer).

Detailed Research Activities:

Tissue Engineering (TE) aims at modifying the clinical practice in the field of regenerative medicine, through a multi-phasic and automatic approach. For instance, multipotent cells obtained by an autologous harvest may be processed and expanded in vitro to amplify their number, seeded into suitable three-dimensional (3D) resorbable biomaterials, and then elaborated in vitro to induce their differentiation and neo-tissue formation, before their implant. Main goals of tissue engineering approach are (i) the identification of the best cellular sources and bio-active molecules, (ii) planning and development of 3D biomaterials with internal structures and chemical compositions favouring the in vivo tissue formation, (iii) the development of specific bioreactor systems for the multipotent cells culture within three-dimensional biomaterials under specific physical stimuli and automatic experimental conditions.
For TE applications, bone marrow stromal cells (BMSC) are typically first expanded in monolayers (2D), in order to overcome their very low fraction among bone marrow cells, and then statically loaded into three-dimensional (3D) porous scaffolds, which prime cell differentiation and provide the template for tissue formation. In all these approaches, the phase of BMSC expansion in 2D is associated not only with biological concerns, such as the loss of cell differentiation capacity with serial passaging, but also with a non-standardized and labor-intensive production of the osteoinductive grafts (e.g., for serial passaging or seeding of the expanded cells into a 3D scaffold). In addition, the static loading of the cells into 3D scaffolds may result in non-uniform distributions, with higher cellular density at the surface layers.
Based on these considerations, our laboratory has been focusing its attention on generating osteoinductive constructs within a 3D culture environment by using a perfusing bioreactor system. In particular, we demostrated that BMSC can be isolated and expanded within 3D ceramic scaffolds by direct loading and culture of bone marrow nucleated cells into the scaffold pores. Moreover, based on the demonstrated efficacy of 3D perfusion systems to improve the efficiency and uniformity of cell seeding within porous 3D scaffolds, we have investigated the effects of dynamic perfusion seeding of bone marrow nucleated cells within 3D ceramics, in terms of the amount and uniformity of bone formed in vivo as compared to static cell loading.
Although perfusion bioreactor systems represent a highly promising tool to engineer osteoinductive grafts, as well as a model to investigate the mechanisms of cell response to flow-induced shear, unraveling a relationship between defined levels of shear and cell response in 3D scaffold-based culture systems is challenged by several factors. For example, the distribution of fluid flow through the porous scaffold microarchitecture is often non-uniform, and the scaffold composition and surface properties, by determining the cell adhesion strength, inherently modulate cell response to shear. Moreover, the effects of shear are likely to depend on the density and spatial organization of cells in the 3D environment, which currently cannot be easily monitored during the time frame of the experiment.
The response of osteogenic cells to shear may be investigated under more controlled conditions in 2D culture configurations, generally consisting of parallel plate flow chambers. These systems have been used to establish that short-term (i.e., 0.5 - 12 hours) exposure of bone derived cells to rather high levels (i.e., 0.5 – 2 dynes/cm2) of flow-induced shear affect cell proliferation and regulate gene expression. However, these results cannot be easily related to typical 3D culture systems, where exposure to flow-induced shear is different in terms of (i) longer duration (up to 1-2 weeks), (ii) lower levels (0.2-1.5.10 N/m2), and (iii) culture substrate (different from tissue-culture treated polystyrene or glass). An objective of our research activity was therefore to investigate the effect of prolonged exposure of human BMSC to flow-induced shear in a 2D culture system, and whether such effect is modulated by the culture substrate (uncoated glass vs glass coated with calcium phosphate). Numerical modeling has also been introduced to verify that the levels of applied shear were spatially uniform within the culture system and in the range typically used in 3D perfusion systems (0.2-1.5*10-3 N/m2). The effects of shear were assessed by analysis of gene expression, matrix deposition, and cytoskeleton organization. We have observed that MSC exposed to fluid flow under 2D bioreactor systems displayed an increased osteogenic commitment, although phenotype changes in response to flow were dependent on the substrate used. These findings highlight the importance of the combination of physical forces and culture substrate in determining the functional state of differentiating osteoblastic cells.
Starting from these considerations, we also aim at identifying genes involved in the mechanism of proliferation and differentiation of human MSC by studying their gene expression profile through the micro-array technique. Advanced technologies and knowledge related to production and treatment of gene expression micro-array distributed data have been implemented, in collaboration with the Bio-Lab laboratory of University of Genova and the genomic unit of ABC. In particular, we aim at analysing the gene expression profile by micro-array, by evaluating the gene expression of BMSC grown under various culture conditions, in order to analyse the effects of different growth conditions (bi- vs. tri-dimensional cultures, static versus dynamic culture) on gene expression with particular regard to genes that are involved in the regulation of cell proliferation and differentiation and stem cell specific traits.
Lastly, we are interested on the identification of the best internal structures and chemical compositions of the 3D scaffolds to be used as bone grafts/substitutes. Besides chemical composition, the other critical parameter to improve the efficiency of biomaterials to be used in bone tissue engineering is the overall structure: density, pore shape, pore size and pore interconnection pathway. Porosity is necessary for the in vivo bone tissue ingrowth since it allows migration and proliferation of osteoblasts and mesenchymal cells, and matrix deposition in the empty spaces. Although macroporosity has a strong impact on osteogenic outcomes, interconnection pathway plays an important role as well. An incomplete pore interconnection could represent a constraint of the overall biological system, limiting blood vessels invasion. A proper blood supply represents the base for bone tissue growth in porous biomaterials.
Therefore, we have tested and compared several osteoconductive porous biomaterials with different internal architecture and chemical composition, by using an established model of in vivo bone formation by exogenously added osteoprogenitor cells. In parallel, we have designed, developed and tested a prototype 3D osteoconductive graft, completely based on an “open-structure” concept, in order to favour vascularisation of the graft and in principle guarantee an unlimited neo-bone tissue ingrowth. This new concept of biomaterials should overcome the intrinsic limits of their internal structure of the most porous materials available.

Applications and Developments:

The main applications of the Bioengineering Laboratory research activity are on generation of 3D tissues to be used as grafts for replacement of damaged organs, and on the in vitro model systems of tissue development and function. In particular, we provide future applications in the planning and development of 3D biomaterials with proper internal structures and chemical compositions, and in the planning and development of specific bioreactor systems for the cell culture within 3D biomaterials under specific physical stimuli and monitored and automatic experimental conditions.

Managed core facilities:

1. 2D and 3D bioreactor systems for cell culture under fluid-dynamic conditions
2. a graphic workstation for computational fluid dynamic simulation.
3. fluorescence optical microscope

Ongoing collaborations:

Our research in the field of Tissue engineering is going on in collaboration with the Laboratory for Tissue Engineering at the University Hospital of Basel (Ivan Martin), Finceramica Faenza s.r.l, DIST department of University of Genoa and the Laboratory of Stem Cells Biology at ABC (Prof. Rodolfo Quarto).

Most recent and significant publications:

Mastrogiacomo, M., S. Scaglione, R. Martinetti, L. Dolcini, F. Beltrame, R. Cancedda, and R. Quarto. Role of scaffold internal structure on in vivo bone formation in macroporous calcium phosphate bioceramics. Biomaterials 2006. Jun;27(17):3230-7.

Beltrame, F., R. Cancedda, B. Canesi, A. Crovace, M. Mastrogiacomo, R. Quarto, S. Scaglione, C. Valastro, and F. Viti. 2005. A simple non invasive computerized method for the assessment of bone repair within osteoconductive porous bioceramic grafts. Biotechnol Bioeng. 92:189-98.

M. Mastrogiacomo, A. Corsi, E. Francioso, M. Di Comite, F. Monetti, S. Scaglione, A. Favia, A. Crovace, P. Bianco, and R. Cancedda, Reconstruction of extensive long-bone defects in sheep using resorbable bioceramics based on Silicon Stabilized Tricalcium Phosphate (Si-TCP). Tissue Eng. 2006 May;12(5):1261-73

S. Scaglione, A. Braccini, D. Wendt, C. Jaquiery, F. Beltrame, R. Quarto, I. Martin, Engineering of osteoinductive grafts by isolation and expansion of ovine bone marrow stromal cells directly on 3D ceramic scaffolds. Biotechnology and Bioengineering, 2006 Jan 5;93(1):181-7.

S. Scaglione, D. Wendt, S. Miggino, A. Papadimitropoulos, M. Fato, R. Quarto and I. Martin Effects of flow-induced shear and calcium-phosphate coating on human bone marrow stromal cells cultured in a defined 2D model system. Journal of Biomedical Material Research Part A, 2007 Oct 29.

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