Controllo molecolare del differenziamento osteoblestico per applicazioni nel campo della rigenerazione del tessuto osseo

The aim of the study was to identify expression signatures unique for specific stages of osteoblast differentiation in order to improve our knowledge of the molecular mechanisms underlying bone repair and regeneration. We performed a microarray analysis on the whole transcriptome of human mesenchymal stem cells (hMSCs) obtained from the femoral canal of patients undergoing hip replacement. By defining different time-points within the differentiation and mineralization phases of hMSCs, temporal gene expression changes were visualised. Importantly, the gene expression of adherent bone marrow mononuclear cells, being the undifferentiated progenitors of bone cells, was used as reference. In addition, only the cultures able to form mineral nodules at the final time-point were considered for the gene expression analyses. To obtain the genes of our interest, we only focused on genes: i) whose expression was significantly upregulated; ii) which are involved in pathways or biological processes relevant to proliferation, differentiation and functions of bone cells; iii) which changed considerably during the different steps of differentiation and/or mineralization. Among the 213 genes identified as differentially expressed by microarray analysis, we selected 65 molecular markers related to specific steps of osteogenic differentiation. These markers are grouped into various gene clusters according to their involvement in processes which play a key role in bone cell biology such as angiogenesis, ossification, cell communication, development and in pathways like TGF beta and Wnt signaling pathways. Taken together, these results allow us to monitor hMSC cultures and to distinguish between different stages of differentiation and mineralization. The signatures represent a useful tool to analyse a broad spectrum of functions of hMSCs cultured on scaffolds, especially when the constructs are conceived for releasing growth factors or other signals to promote bone regeneration. Morover, this work will enhance our understanding of bone development and will enable us to recognize molecular defects that compromise normal bone function as occurs in pathological conditions.

Valutazione del ruolo dell'espressione di IRS-1 nel differenziamento osteoblastico di cellule di osteosarcoma ed MSCs

L’osteosarcoma (OS) è il tumore primitivo dell’osso più comune in età pediatrica e adolescenziale. L’OS è stato recentemente riconsiderato come una patologia da de-differenziamento, legata all’interruzione del processo cui vanno incontro i precursori osteoblastici, quali le cellule staminali mesenchimali (MSCs), per trasformarsi in osteoblasti maturi. Il sistema IGF è coinvolto nella regolazione della proliferazione e del differenziamento di cellule di OS. IRS-1 è un mediatore critico di tale via di segnalazione e il suo livello di espressione modula il differenziamento di cellule ematopoietiche. Lo scopo di questa tesi è stato quello di definire il ruolo di IRS-1 nel differenziamento osteoblastico di MSCs e cellule di OS. Il potenziale differenziativo di cellule di OS umano e murino e di MSCs derivate da midollo osseo è stato valutato tramite Alizarin Red staining e Real Time-PCR. Dai dati ottenuti è emerso come i livelli di espressione di IRS-1 diminuiscano durante il differenziamento osteoblastico. Conseguentemente, i livelli di espressione di IRS-1 sono stati manipolati utilizzando shRNA per down-regolare l’espressione della proteina o un plasmide per sovra-esprimerla. Sia la down-regolazione sia la sovra-espressione di IRS-1 hanno inibito il differenziamento osteoblastico delle linee cellulari considerate. Allo scopo di valutare il contributo di IRS-1 nella via di segnalazione di IGF-1R è stato utilizzato l’inibitore di tale recettore, αIR-3. Anche in questo caso è stata osservata una riduzione della capacità differenziativa. L’inibitore del proteasoma MG-132 ha portato ad un aumento dei livelli di IRS-1, portando nuovamente all’inibizione del differenziamento osteoblastico e suggerendo che l’ubiquitinazione di questa proteina potrebbe avere un ruolo importante nel mantenimento di appropriati livelli di espressione di IRS-1. I risultati ottenuti indicano la criticità dei livelli di espressione di IRS-1 nella determinazione della capacità differenziativa sia di cellule di OS umano e murino, sia delle MSCs.

Development of newly conceived biomimetic nano-structured biomaterials as scaffolds for bone and osteochondral regeneration

The present research thesis was focused on the development of new biomaterials and devices for application in regenerative medicine, particularly in the repair/regeneration of bone and osteochondral regions affected by degenerative diseases such as Osteoarthritis and Osteoporosis or serious traumas. More specifically, the work was focused on the synthesis and physico-chemical-morphological characterization of: i) a new superparamagnetic apatite phase; ii) new biomimetic superparamagnetic bone and osteochondral scaffolds; iii) new bioactive bone cements for regenerative vertebroplasty. The new bio-devices were designed to exhibit high biomimicry with hard human tissues and with functionality promoting faster tissue repair and improved texturing. In particular, recent trends in tissue regeneration indicate magnetism as a new tool to stimulate cells towards tissue formation and organization; in this perspective a new superparamagnetic apatite was synthesized by doping apatite lattice with di-and trivalent iron ions during synthesis. This finding was the pin to synthesize newly conceived superparamagnetic bone and osteochondral scaffolds by reproducing in laboratory the biological processes yielding the formation of new bone, i.e. the self-assembly/organization of collagen fibrils and heterogeneous nucleation of nanosized, ionically substituted apatite mimicking the mineral part of bone. The new scaffolds can be magnetically switched on/off and function as workstations guiding fast tissue regeneration by minimally invasive and more efficient approaches. Moreover, in the view of specific treatments for patients affected by osteoporosis or traumas involving vertebrae weakening or fracture, the present work was also dedicated to the development of new self-setting injectable pastes based on strontium-substituted calcium phosphates, able to harden in vivo and transform into strontium-substituted hydroxyapatite. The addition of strontium may provide an anti-osteoporotic effect, aiding to restore the physiologic bone turnover. The ceramic-based paste was also added with bio-polymers, able to be progressively resorbed thus creating additional porosity in the cement body that favour cell colonization and osseointegration.

Development of musculoskeletal models for the design and the pre-clinical validation of hip resurfacing prosthesis

Background. The surgical treatment of dysfunctional hips is a severe condition for the patient and a costly therapy for the public health. Hip resurfacing techniques seem to hold the promise of various advantages over traditional THR, with particular attention to young and active patients. Although the lesson provided in the past by many branches of engineering is that success in designing competitive products can be achieved only by predicting the possible scenario of failure, to date the understanding of the implant quality is poorly pre-clinically addressed. Thus revision is the only delayed and reliable end point for assessment. The aim of the present work was to model the musculoskeletal system so as to develop a protocol for predicting failure of hip resurfacing prosthesis. Methods. Preliminary studies validated the technique for the generation of subject specific finite element (FE) models of long bones from Computed Thomography data. The proposed protocol consisted in the numerical analysis of the prosthesis biomechanics by deterministic and statistic studies so as to assess the risk of biomechanical failure on the different operative conditions the implant might face in a population of interest during various activities of daily living. Physiological conditions were defined including the variability of the anatomy, bone densitometry, surgery uncertainties and published boundary conditions at the hip. The protocol was tested by analysing a successful design on the market and a new prototype of a resurfacing prosthesis. Results. The intrinsic accuracy of models on bone stress predictions (RMSE < 10%) was aligned to the current state of the art in this field. The accuracy of prediction on the bone-prosthesis contact mechanics was also excellent (< 0.001 mm). The sensitivity of models prediction to uncertainties on modelling parameter was found below 8.4%. The analysis of the successful design resulted in a very good agreement with published retrospective studies. The geometry optimisation of the new prototype lead to a final design with a low risk of failure. The statistical analysis confirmed the minimal risk of the optimised design over the entire population of interest. The performances of the optimised design showed a significant improvement with respect to the first prototype (+35%). Limitations. On the authors opinion the major limitation of this study is on boundary conditions. The muscular forces and the hip joint reaction were derived from the few data available in the literature, which can be considered significant but hardly representative of the entire variability of boundary conditions the implant might face over the patients population. This moved the focus of the research on modelling the musculoskeletal system; the ongoing activity is to develop subject-specific musculoskeletal models of the lower limb from medical images. Conclusions. The developed protocol was able to accurately predict known clinical outcomes when applied to a well-established device and, to support the design optimisation phase providing important information on critical characteristics of the patients when applied to a new prosthesis. The presented approach does have a relevant generality that would allow the extension of the protocol to a large set of orthopaedic scenarios with minor changes. Hence, a failure mode analysis criterion can be considered a suitable tool in developing new orthopaedic devices.

An alternative B cell development program

Two major types of B cells, the antibody-producing cells of the immune system, are classically distinguished in the spleen: marginal zone (MZ) and follicular (FO). In addition, FO B cells are subdivided into FO I and FO II cells, based on the amount of surface IgM. MZ B cells, which surround the splenic follicles, rapidly produce IgM in response to blood-borne pathogens without T cell help, while T cell-dependent production of high affinity, isotype-switched antibodies is ascribed to FO I cells. The significance of FO II cells and the mechanism underlying B cell fate choices are unclear. We showed that FO II cells express more Sca1 than FO I cells and originate from a distinct B cell development program, marked by high expression of Sca1. MZ B cells can derive from the “canonical” Sca1lo pathways, as well as from the Sca1hi program, although the Sca1hi program shows a stronger MZ bias than the Sca1lo program, and extensive phenotypic plasticity exists between MZ and FO II, but not between MZ and FO I cells. The Sca1hi program is induced by hematopoietic stress and generates B cells with an Igλ-enriched repertoire. In aged mice, the canonical B cell development pathway is impaired, while the Sca1hi program is increased. Furthermore, we showed that a population of unknown function, defined as Lin-c-kit+Sca1+ (LSK-), contains early lymphoid precursors, with primarily B cell potential in vivo. Our data suggest that LSK- cells may represent a distinct precursor for the Sca1hi program in the bone marrow.

Synthesis and characterization of implants for bone substitutions made of biomedical apatites containing silicon

In the last decades the development of bone substitutes characterized by a superior biomimetism has become of particular interest, owing to the increasing economic and societal impact of the bone diseases. In the present work of research the development of bone substitutes characterized by improved biomimetism, has been faced in a chemical, structural and morphological perspective. From a chemical point of view, it has been developed the synthesis of hydroxyapatite powders, exhibiting multiple ionic substitutions in both cationic and anionic sites, so to simulate the chemical composition of the natural bone. Particular emphasis has been given to the effect of silicon on the chemical-physical and solubility properties of the obtained hydroxyapatites. From a structural point of view, it has been developed the synthesis of ceramic composite materials, based on hydroxyapatite and calcium silicates, employed both as a reinforcing phase, to raise the mechanical strength of the composite compared to hydroxyapatite, and as a bioactive phase, able to increase the bioactivity properties of the whole ceramic. Finally the unique morphological features of the bone were mimicked by taking inspiration by Nature, so that native wood structures were treated in chemical and thermal way to obtain hydroxyapatite porous materials characterized by the same morphology as the native wood. The results obtained in the present work were positive in all the three different areas of investigation, so to cover the three different aspects of biomimetism, chemical, structural and morphological. Anyway, only at the convergence of the three different fields it is possible to find out the best solutions to develop the ideal bone-like scaffold. Thus, the future activity should be devoted to solve the problems at the borderline between the different research lines, which hamper this convergence and in consequence, the achievement of a bone scaffold able to mimic the various aspects exhibited by the bone tissue

Evaluation of bone strength: microtomographic tecniques

This thesis is a part of a larger study about the characterization of mechanical and histomorphometrical properties of bone. The main objects of this study were the bone tissue properties and its resistance to mechanical loads. Moreover, the knowledge about the equipment selected to carry out the analyses, the micro-computed tomography (micro-CT), was improved. Particular attention was given to the reliability over time of the measuring instrument. In order to understand the main characteristics of bone mechanical properties a study of the skeletal, the bones of which it is composed and biological principles that drive their formation and remodelling, was necessary. This study has led to the definition of two macro-classes describing the main components responsible for the resistance to fracture of bone: quantity and quality of bone. The study of bone quantity is the current clinical standard measure for so-called bone densitometry, and research studies have amply demonstrated that the amount of tissue is correlated with its mechanical properties of elasticity and fracture. However, the models presented in the literature, including information on the mere quantity of tissue, have often been limited in describing the mechanical behaviour. Recent investigations have underlined that also the bone-structure and the tissue-mineralization play an important role in the mechanical characterization of bone tissue. For this reason in this thesis the class defined as bone quality was mainly studied, splitting it into two sub-classes of bone structure and tissue quality. A study on bone structure was designed to identify which structural parameters, among the several presented in the literature, could be integrated with the information about quantity, in order to better describe the mechanical properties of bone. In this way, it was also possible to analyse the iteration between structure and function. It has been known for long that bone tissue is capable of remodeling and changing its internal structure according to loads, but the dynamics of these changes are still being analysed. This part of the study was aimed to identify the parameters that could quantify the structural changes of bone tissue during the development of a given disease: osteoarthritis. A study on tissue quality would have to be divided into different classes, which would require a scale of analysis not suitable for the micro-CT. For this reason the study was focused only on the mineralization of the tissue, highlighting the difference between bone density and tissue density, working in a context where there is still an ongoing scientific debate.

Biodegradable systems for the development of functional materials

This PhD work was aimed to design, develop, and characterize gelatin-based scaffolds, for the repair of defects in the muscle-skeletal system. Gelatin is a biopolymer widely used for pharmaceutical and medical applications, thanks to its biodegradability and biocompatibility. It is obtained from collagen via thermal denaturation or chemical-physical degradation. Despite its high potential as biomaterial, gelatin exhibits poor mechanical properties and a low resistance in aqueous environment. Crosslinking treatment and enrichment with reinforcement materials are thus required for biomedical applications. In this work, gelatin based scaffolds were prepared following three different strategies: films were prepared through the solvent casting method, electrospinning technique was applied for the preparation of porous mats, and 3D porous scaffolds were prepared through freeze-drying. The results obtained on films put into evidence the influence of pH, crosslinking and reinforcement with montmorillonite (MMT), on the structure, stability and mechanical properties of gelatin and MMT/gelatin composites. The information acquired on the effect of crosslinking in different conditions was utilized to optimize the preparation procedure of electrospun and freeze-dried scaffolds. A successful method was developed to prepare gelatin nanofibrous scaffolds electrospun from acetic acid/water solution and stabilized with a non-toxic crosslinking agent, genipin, able to preserve their original morphology after exposure to water. Moreover, the co-electrospinning technique was used to prepare nanofibrous scaffolds at variable content of gelatin and polylactic acid. Preliminary in vitro tests indicated that the scaffolds are suitable for cartilage tissue engineering, and that their potential applications can be extended to cartilage-bone interface tissue engineering. Finally, 3D porous gelatin scaffolds, enriched with calcium phosphate, were prepared with the freeze-drying method. The results indicated that the crystallinity of the inorganic phase influences porosity, interconnectivity and mechanical properties. Preliminary in vitro tests show good osteoblast response in terms of proliferation and adhesion on all the scaffolds.

Pre-clinical and clinical development of leukemia stem cell inhibitors in acute leukemias

In Leukemias, recent developments have demonstrated that the Hedgehog pathway plays a key-role in the peculiar ability of self renewal of leukemia stem cells. The aim of this research activity was to investigate, through a first in man, Phase I, open label, clinical trial, the role and the impact, mainly in terms of safety profile, adverse events and pharmacokinetics, of a Sonic Hedgehog inhibitor compound on a population of heavely pretreated patients affected by AML, CML, MF, or MDS, resistant or refractory to standard chemotherapy. Thirty-five patients have been enrolled. The drug was administered orally, in 28 days cycles, without rest periods. The compound showed a good safety profile. The half life was of 17-35 hours, justifying the daily administration. Significant signs of activity, in terms of reduction of bone marrow blast cell amount were seen in most of the patients enrolled. Interestingly, correlative biological studies demonstrated that, comparing the gene expression profyiling signature of separated CD34+ cells before and after one cycle of treatment, the most variably expressed genes were involved in the Hh pathway. Moreover, we observed that many genes involved in MDR (multidrug resistance)were significantly down regulated after treatment. These data might lead to future clinical trials based on combinatory approaches, including, for instance, Hh inhibitors and conventional chemotherapy.

Development of glass-ceramics from combination of industrial wastes together with boron mining waste

The utilization of borate mineral wastes with glass-ceramic technology was first time studied and primarily not investigated combinations of wastes were incorporated into the research. These wastes consist of; soda lime silica glass, meat bone and meal ash and fly ash. In order to investigate possible and relevant application areas in ceramics, kaolin clay, an essential raw material for ceramic industry was also employed in some studied compositions. As a result, three different glass-ceramic articles obtained by using powder sintering method via individual sintering processes. Light weight micro porous glass-ceramic from borate mining waste, meat bone and meal ash and kaolin clay was developed. In some compositions in related study, soda lime silica glass waste was used as an additive providing lightweight structure with a density below 0.45 g/cm3 and a crushing strength of 1.8±0.1 MPa. In another study within the research, compositions respecting the B2O3–P2O5–SiO2 glass-ceramic ternary system were prepared from; borate wastes, meat bone and meal ash and soda lime silica glass waste and sintered up to 950ºC. Low porous, highly crystallized glass-ceramic structures with density ranging between 1.8 ± 0,7 to 2.0 ± 0,3 g/cm3 and tensile strength ranging between 8,0 ± 2 to 15,0 ± 0,5 MPa were achieved. Lastly, diopside - wollastonite (SiO2-Al2O3-CaO )glass-ceramics from borate wastes, fly ash and soda lime silica glass waste were successfully obtained with controlled rapid sintering between 950 and 1050ºC. The wollastonite and diopside crystal sizes were improved by adopting varied combinations of formulations and heating rates. The properties of the obtained materials show; the articles with a uniform pore structure could be useful for thermal and acoustic insulations and can be embedded in lightweight concrete where low porous glass-ceramics can be employed as building blocks or additive in cement and ceramic industries.