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Regenerative Medicine

Introduction

New strategies to promote musculoskeletal tissue healing by stimulating intrinsic properties of mesenchymal cells, including growth factors and cytokines release, is a major research issue, recognising that the regenerative potential of mesenchymal cells is modulated by micro-and nanostructured substrates mimicking the extracellular matrix, too.

Cellular models

For the development of cell therapies, mesenchymal stromal cells (MSC) are ideal for the regeneration of tissues thanks to their self renewal and multipotency properties, as well as trophic, or paracrine, activity. In order to simulate the MSC behavior in different pathophysiological conditions, they are grown under standard conditions, or selective microenvironments, such as relative hypoxia, 3D pellet, serum deprivation, etc. Also the regenerative potential of circulating or late-adherent bone marrow-derived MSC is under investigation.Figure 1 - MSC, expression of alkaline phosphatase

Endothelial cells from umbilical vein (HUVEC) are used to study angiogenesis and to analyze their activities in tissue regeneration, i.e. expression of growth factors and cytokines, chemotaxis, etc.Figure 2 - HUVEC expression of Ve-cadherin

Osteoclast precursors are obtained from circulation and cultured in order to study bone remodeling. Mature osteoclasts are multi-nucleated, express typical mature osteoclast markers, and are able to adhere on the bone substrate and to degrade the mineral and organic components. Following interactions with different substrates, either natural or synthetic, the presence of adhesion structures and the cytoskeletal pattern, as well as the ability to resorb the substrate, to degrade collagen type I, and to express specific markers are evaluated.

Figure 3 - Osteoclasts: different organization of the cytoskeleton on poly-dimethyl siloxane (A), glass (B), oxo (C)

Cell-substrate interaction

Surface charge and stiffness of the substrate dictate cell adhesion step and subsequent proliferation and differentiation. In this respect, cell adhesion, with particular attentionto the role of the pericellular matrix is a matter of research. Innovative bone substitutes with micro / macroporosity, bioactivity, biodegradability, and surface functionalization by nanostructuring, plasma treatment or peptide binding, are investigated. Three studies focused on these issues have been recently conducted. Under the Nanobiocom project (EC-FP6) a polylactide-based resorbable composite added with carbon nanotubes, microhydroxyapatite and BMP-2 has been synthesised and characterized. The project SmartCaP (EC-FP6) has developed a degradable injectable composite, based on calcium phosphate, gelatin and soy, to fill bone defects. The interactions of MSC and recombinant peptides derived from elastin are studied in the framework of a national MIUR-PRIN project.

Growth factors

The activity of growth factors is a prerequisite for ensuring the efficacy of regenerative therapies. The common strategy is based on a combination of platelet gel-derived growth factors (GF) and bone marrow-derived osteogenic precursors. Since GF proportion is variable depending on the preparation and storage of platelet concentrate (PRP), the pro-osteogenic activity of differently prepared PRP is assayed.

Cellular and molecular biology of bone

The gene expression profile of bone marrow-derived osteo-induced MSC has been identified under the Nanobiocom project (EC-FP6). This database is used to study MSC on 2D substrates, as well as gene expression in bone diseases such as congenital pseudarthrosis and osteogenesis imperfecta. To better understand the fine regulation of MSC osteogenic differentiation, post-transcriptional regulation by microRNAs (miRNAs) is investigated. The role of insulin, insulin-like growth factors and their receptors in skeletal homeostasis are studied to understand the mutual and bidirectional influence between bone and energy metabolism during bone remodeling.

Clinical Studies

Launched in January 2010 under EC-FP7, the REBORNE project aims to demonstrate the safety and efficacy of expanded autologous stem cells combined with biomaterials for bone repair in nonunions and osteonecrosis. Predictive assays of osteoinductive capacity of a) platelet gel for orthopedic applications, and b) serum from patients with nonunions of long bones are under validation.