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, and we left 6 untreated lesioned bone. 8 weeks after surgery animals were sacrificed and the tibia explanted. A macroscopic analysis showed no bone resorption, no abnormal bone callous formation, no fractures, infection or inflammatory reactions, and all the bone defects were completely filled without any significant differences among the four groups. Interestingly, in the presence of scaffold seeded with rbASCs, histology and immunohistochemistry showed a new bone tissue more mature and similar to the native bone. These data have also been confirmed by biomechanical tests: indeed, the mechanical properties of the bone defect treated by rbASCs-HA were improved, suggesting that these constructs bore mechanical loading with an increase in stiffness of 19.8% and in hardness of 31.6% respect to just HA treated group, indicating that the bioconstructs made out of autologous rbASCs and hydroxyapatite might ameliorate the treatment for large bone defects. We would suggest the use of ASCs as a safe cellular therapy in future clinical applications where a large bone defect needs to be treated. These promising results on small size animals allow us to plan a new study on large size animals such as minipigs. However, before moving to the clinic, we know that there are several important aspects that need to be faced regarding safeness and the features of the candidate patients: 1. may the “quality” of hASCs be affected by the donor’s physiological or pathological conditions? 2. may the use of pharmacological treatment enhance cellular plasticity of multipotent cells? 3. may the use of immunoselected hASCs ameliorate tissue regeneration in the field of muscle-skeletal? We have addressed some of these aspects, comparing different populations of hASCs from subcutaneous adipose tissue of healthy-young-female donors (hASCs<35 y/o, n=12, mean age 31±4 years, BMI=23.5±1.6), and from middle-age ones (hASCs>45 y/o n=14, mean age 56±7 years, mean BMI=28.4±1.8). The cellular yield of hASCs derived from older donors was 2.5 fold greater than the one of hASCs<35 y/o, whereas hASCs from younger donors were more clonogenic than hASCs isolated from older ones, with an increase of 129%. No significant differences were observed looking at their immunophenotype. When hASCs were induced to differentiate into cells of the adipogenic and osteogenic lineages, the donor’s age did not affect their adipogenic differentiation, whereas the osteogenic one was significantly affected by age both in the absence and in the presence of three-dimensional scaffolds, showing a decreased ALP basal levels of about 10-fold in hASCs>45 y/o respect to hASCs<35 y/o. These results seems to indicate that ASCs from different donors could behave differently. Trying to overcome this aspect we have used different approaches, and we have studied if Reversine, a synthetic purine already known to increase plasticity of terminally differentiated cells, might improve the differentiation ability of hASCs. 72 hours treatment with 50 nM Reversine induced hASCs to differentiate into osteoblast like-cells (+45% of alkaline phosphatase activity), smooth muscle cells (+89% of α-actin expression) and skeletal muscle cells (myotubes formation) compared to control hASCs. Moreover, since it is known that CD34 and L-NGFR positive cells define a subset of high proliferative and multipotent MSCs, we have immunoselected, these progenitor cells from hASC populations. In contrast to the whole population, the immunoseparated fractions maintained their undifferentiate state and their ability to differentiate much longer during culture. We have shown that both CD34+ and L-NGFR+ hASCs can be used as alternative candidates for tissue engineering and regenerative medicine applications. In particular, due to the improved ability of L-NGFR positive cells to adipo- and chondro-differentiate, they appear an ideal tool in reconstructive plastic surgery and cartilage regeneration. From our data, and the ones from researchers in other fields, we believe that in the near future adipose-derived stem cells might be considered a safe tool in regenerative medicine. Furthermore, to improve this “cellular therapy”, we could either pre-treat ASCs with molecules, such as drugs and/or siRNAs known to affect specific differentiation pathways, or by selecting subpopulations of progenitor cells which may be used as allogenic implants. Next step will be to confirm our in vivo data in a large size animal model such as minipig, and then to test if pre-treated cells or selected population might be used in an autologous and allogenic small size animal model.
ADIPOSE-DERIVED STEM CELLS (ASCS) FOR FUTURE CELLULAR THERAPIES IN MUSCLE-SKELETAL TISSUES REGENERATION / E. Arrigoni ; tutor: A.T. Brini ; coordinatore: A.E. Panerai. - Milano : Università degli studi di Milano. Universita' degli Studi di Milano, 2012 Feb 03. ((24. ciclo, Anno Accademico 2011.
|Titolo:||ADIPOSE-DERIVED STEM CELLS (ASCS) FOR FUTURE CELLULAR THERAPIES IN MUSCLE-SKELETAL TISSUES REGENERATION|
|Supervisori e coordinatori interni:||PANERAI, ALBERTO EMILIO|
|Data di pubblicazione:||3-feb-2012|
|Parole Chiave:||adipose-derived stem cells ; animal models ; critical bone defect; multidifferentiative potential ; tissue engineering and egenerative medicine ; cellular plasticity ; reversine|
|Settore Scientifico Disciplinare:||Settore BIO/14 - Farmacologia|
|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. - Milano : Università degli studi di Milano. Universita' degli Studi di Milano, 2012 Feb 03. ((24. ciclo, Anno Accademico 2011.|
|Digital Object Identifier (DOI):||http://dx.doi.org/10.13130/arrigoni-elena_phd2012-02-03|
|Appare nelle tipologie:||Tesi di dottorato|