ADIPOSE-DERIVED STEM CELLS (ASCS) FOR FUTURE CELLULAR THERAPIES IN MUSCLE-SKELETAL TISSUES REGENERATION
Tesi di Dottorato
Data di Pubblicazione:
2012
Citazione:
ADIPOSE-DERIVED STEM CELLS (ASCS) FOR FUTURE CELLULAR THERAPIES IN MUSCLE-SKELETAL TISSUES REGENERATION / E. Arrigoni ; tutor: A.T. Brini ; coordinatore: A.E. Panerai. Universita' degli Studi di Milano, 2012 Feb 03. 24. ciclo, Anno Accademico 2011. [10.13130/arrigoni-elena_phd2012-02-03].
Abstract:
Every year several patients have to deal with bone tissue loss due to trauma or diseases. Bone tissue engineering aims to restore or repair musculoskeletal disorders through the development of bio-substitutes that require the use of cells and scaffolds which should possess both adequate mechanical properties and interconnecting pores to allow cellular infiltration, graft integration and vascularization. The ideal cell for tissue engineering should possess a potential plasticity with the ability to functionally repair the damaged tissue, and it should be available in large amount. Mesenchymal stem cells (MSCs) are present in many adult tissues, and adipose tissue represents an attractive source of MSCs for researchers and clinicians of nearly all medical specialties. Adipose-derived stem cells (ASCs) are similar to MSCs isolated from bone marrow, placenta, and umbilical cord blood in morphology, immunophenotype, and differentiation ability, and they represent a promising approach of bone regeneration. Additional features of ASCs are their immunoregolatory and anti-inflammatory properties both in vivo and in vitro and their low immunogenicity.
Since several years our laboratory is studying mesenchymal stem cells isolated from human and animal adipose tissues. Human ASCs (hASCs) have been characterized by their immunophenotype, their self-renewal potential, and they have been induced to differentiate towards adipogenic, osteogenic and chondrogenic lineages. The ability of hASCs to grow in the presence of several scaffolds has also been tested. hASCs adhered to the surface of tested biomaterials, filling the pores and forming a 3D web-like structure, allowing these progenitor cells to osteo-differentiate more efficiently respect to cells maintained on polystyrene. Since our interest was to regenerate muscle-skeletal defects by ASCs in pre-clinical models, we first studied ASCs isolated from adipose tissue of rat (rASCs), rabbit (rbASCs) and pig (pASCs), considered good models in the orthopaedic field. We have shown that animal ASCs behaved similarly to the human ones, and, in collaboration with the Faculty of Veterinary Medicine of University of Milan and the IRCCS Galeazzi Orthopaedic Institute of Milan, we have tested the ability of autologous ASCs to regenerate a full-thickness critical-size bone defect in rabbits. The experimental study was conducted on the tibiae of 12 New Zealand rabbits, and from 6 rabbits out of 12 we have collected adipose tissue from the interscapular region. We have isolated 2.8x105±1.9x105 rbASCs per ml of raw tissue, and after 3-4 days in culture the cells showed the typical fibroblast-like morphology. One week later, all the 6 cellular populations started to steadily proliferate, and they generated fibroblast (CFU-F) and osteoblast (CFU-O) colonies, highlighting the presence of osteogenic progenitors. Indeed, when rbASCs were induced to osteo-differentiate, either after 7 and 14 days, we have observed an up-regulation of specific osteogenic markers, such as alkaline phosphatase (ALP, +28.9%), collagen (+105.9%) and extracellular calcified matrix (+168.1%), compared to undifferentiated cells. In parallel, testing HA, the scaffold selected for the in vivo experiment, we found that rbASCs were osteoinduced; indeed the presence of HA granules increased per se the amount of collagen production (+48.2%).
1.5x106 undifferentiated rbASCs were seeded on custom-made HA disks (8 mm Ø x 4 mm ↕), and the day after, each bioconstruct was implanted into the lesion created in the tibia of each rabbit. We had an additional experimental group of defects where the same number of rbASCs were inserted in the lesion as a semi-liquid suspension; moreover, as controls, we treated 6 lesioned tibia with just the scaffolds,
Tipologia IRIS:
Tesi di dottorato
Keywords:
adipose-derived stem cells ; animal models ; critical bone defect; multidifferentiative potential ; tissue engineering and egenerative medicine ; cellular plasticity ; reversine
Elenco autori:
E. Arrigoni
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